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Methane production and productivity
changes associated with defaunation in
ruminants
By Son Hung Nguyen
Bachelor of Animal and Veterinary Science, National University of Agriculture, Vietnam
Master of Animal Science and Management, University of Queensland, Australia
A thesis submitted for the degree of Doctor of Philosophy of the
University of New England
June 2016
School of Environmental and Rural Science
Faculty of Arts and Science
i
Executive Summary
With increasing world population, global demand for a secure and growing food
supply challenges the livestock producers of today to increase output of milk and
meat while reducing the environmental impact of animal production. This thesis
reports a review of literature and targeted new research assessing the consequences
of eliminating rumen protozoa (defaunation) on the performance, digestive function
and emissions of the greenhouse gas methane, by livestock.
Comparative studies of rumen fermentation and animal growth were
conducted in growing Merino lambs, crossbred sheep and Brahman cattle. In
these studies ruminants were defaunated using coconut oil distillate to
suppress protozoa then dosed with sodium 1-(2-sulfonatooxyethoxy)
dodecane in a protocol that suppressed feed intake for an average of 10 days
but had no detrimental effects on animal health.
Reflecting the diversity in published literature, these studies found
inconsistent effects of defaunation on volatile fatty acid (VFA) concentrations
and proportions. Averaged over all experiments conducted, defaunation was
associated with a small (5%) reduction in total VFA concentration and an
increase (5%) in the ratio of acetate to propionate in the rumen.
While effects on VFA were not consistent, an average 30% reduction in
rumen ammonia concentration and a 16% increase in microbial crude protein
outflow (estimated by allantoin excretion) were apparent, suggesting
substantial differences in the ruminal degradation and outflow of protein due
to defaunation. These changes were associated with an 18% increase in
average daily gain (ADG), but surprisingly no increase in wool growth rate.
Defaunation was associated with a lower enteric methane emission (average
20% reduction) compared to faunated ruminants, with the first studies of
daily methane production (DMP) ever made while grazing, made using
GreenFeed Emission Monitoring (GEM) units, confirming a 3% lower DMP
ii
(non-significant; P > 0.05) and a 9% lower methane yield (MY; CH4/kg DMI;
P = 0.06) in defaunated sheep.
Protozoa affected the rumen response to nitrate, with the nitrate induced
reduction in MY being 29% greater in faunated compared to defaunated
lambs.
With dietary coconut oil, no interaction with defaunation was apparent with
both coconut oil and defaunation significantly reducing DMP and MY in
cattle.
While defaunation tended to increase average daily gain and reduced enteric
methane emissions in cattle by 10%, establishing defaunated cattle proved
difficult and is a major constraint to expanding defaunation into commercial
herds.
Assessment of the distribution of protozoa in the forestomaches showed that
the number of entodiniomorph protozoa attached to the ‘leaves’ of the bovine
omasum was at least as great as the number attached to the entire surface of
the rumen, though all tissue-attached populations are far fewer than the
population in the rumen fluid.
It is concluded that defaunation alone or in combination with dietary
supplements of nitrate is effective in decreasing methane emissions, while
increasing microbial protein supply and ADG. Commercial implementation
of defaunation for cattle will not be able to rely on addition of surfactants to
the rumen and it is suggested a bioactive compound distributed through the
blood may be needed to remove protozoa residing in the omasum.
In the 6 experimental chapters reporting the research that has been (Chapters 2, 4, 5,
6 and 7) or will be (Chapter 3) submitted for publication, all definitions of
abbreviations have been provided anew in each chapter as required for
publication. Despite some chapters already been published, minor grammatical and
textual improvements have been made (eg. Such as numbering of tables and figs) to
improve the clarity and uniformity of these manuscripts when considered within the
context of this thesis.
iii
Declaration
I declare that the work presented in this thesis is, to the best of my knowledge and
belief, original and my own work, except as acknowledged in the text, that it
accurately and truthfully reflects the information gathered during the research
program I have undertaken, and that the material has not been submitted, either in
whole or in part, for a degree at this or any other university.
Son Hung Nguyen
14th
June, 2016
iv
Acknowledgements
I would firstly like to sincerely thank my principal supervisor, Prof. Roger Hegarty,
for his patience and advice during the time I have worked on my research project.
His advice and encouragement have helped me approach new knowledge and guide
my prospective research. I would also like to thank Dr Lily Li, Dr Gareth Kelly and
Prof. John Nolan who have been supervisors and/or mentors to me during my study.
Their friendly and helpful support is greatly appreciated.
I would like to thank Mr. Graeme Bremner, Mr. Andrew Blakely and Mrs. Jennie
Hegarty who have been friend and assistants to me for almost three years. I also
would like to thank my PhD fellows I. De Barbieri, J. Velazco and V. de Raphélis-
Soissan. Their assistance in helping me collecting data is greatly appreciated. Thank
you to all staff at the Department of Animal Science, the University of New England,
the support that all of them put into my study has made my job much easier, is
gratefully acknowledged.
Most importantly, I would like to acknowledge and sincerely thank my family who
provided and encouraged me to get to this point. Firstly to my wife, who has been an
enormous support and has raised our gorgeous daughters when I have been away.
Secondly, I would like to thank my parents and parents-in-law for their love and
support they have provided my wife and my daughters for more than three years.
Last but not least, this PhD was supported by the University of New England, DVCR
International Fee Scholarship and by Vietnam International Education Development.
Without these supports, these studies would not have happened.
v
Table of Content
Executive Summary i
Declaration iii
Acknowledgements iv
Table of Content v
List of Figures x
List of Tables xi
List of abbreviations xiii
Chapter 1 1
Review of the literature 1
1.1 Overview 1
1.1.1 The making of enteric methane 2
1.1.2 The management of enteric methane 4
1.2 The rumen protozoa and classification 6
1.3 Role of protozoa in ruminant nutrition 9
1.3.1 Plant cell wall digestion 10
1.3.2 Carbohydrate and starch digestion 11
1.3.3 Protein digestion and protozoal synthesis in the rumen 12
1.3.4 Ruminal lipid metabolism 13
1.4 Factors affecting protozoal population densities in the rumen 14
1.4.1 Diet composition 14
1.4.2 Dietary fatty acid supplement 15
1.4.3 Frequency of feeding 16
1.5 Defaunating the rumen 16
1.5.1 Isolation of young ruminants after birth 17
1.5.2 Chemical drenching methods 18
1.5.3 Effect of defaunation on extent of ruminal fermentation 19
1.5.4 Effect of defaunation on ruminal volatile fatty acids 20
1.5.5 Effect of defaunation on animal performance 22
1.5.6 Effect of defaunation on enteric methane production 23
1.6 Effects of oils or nitrate on rumen fermentation and methane emissions 25
1.6.1 Oils 26
1.6.2 Dietary nitrate 27
1.7 Hypothesis of the research 28
vi
Chapter 2 31
Methane emissions, ruminal characteristics and nitrogen utilisation changes after
refaunation of protozoa-free sheep 31
Abstract 32
2.1 Introduction 33
2.2 Materials and methods 34
2.2.1 Preparation of defaunated animals 34
2.2.2 Experiment 1 37
2.2.3 Methane measurement by GreenFeed Emission Monitoring units 38
2.2.4 Experiment 2 39
2.2.5 Estimation of reticulo-rumen weight, gas proportion and carcass composition 40
2.2.6 Nitrogen digestibility, energy utilisation, and microbial protein outflow 40
2.2.7 Methane measurement by respiration chambers 41
2.2.8 Statistical analyses 42
2.3 Results 43
2.3.1 Experiment 1 43
2.3.2 Experiment 2 46
2.4 Discussion 50
2.4.1 Protozoal population in refaunated sheep after inoculation 50
2.4.2 Ruminal fermentation and microbial protein outflow 51
2.4.3 Methane emissions 53
2.4.4 Reticulo-rumen weight and carcass composition 54
2.4.5 Whole-tract dry matter digestibility, nitrogen and energy utilisation 55
2.5 Conclusion 57
Chapter 3 61
Methane emissions and productivity of defaunated and refaunated sheep while
grazing 61
Abstract 62
3.1 Introduction 63
3.2 Materials and methods 63
3.2.1 Animals and experimental procedures 63
3.2.2 Estimation of pasture green dry matter 65
3.2.3 Predicted dry matter intake and dry matter digestibility 66
3.2.4 Methane and carbon dioxide measurement by Greenfeed Emission Monitoring units 69
3.2.5 Analytical procedures 71
3.2.6 Statistical analyses 72
3.3 Results 73
3.3.1 Pastures 73
3.3.2 Ruminal fermentation and methane production 73
vii
3.3.3 Dry matter intake, liveweight gain and wool production 74
3.4 Discussion 76
3.4.1 Animal productivity 76
3.4.2 Rumen fermentation and daily methane production 78
3.5 Conclusion 80
Chapter 4 83
Use of dietary nitrate to increase productivity and reduce methane production of
defaunated and faunated lambs consuming protein deficient chaff 83
Abstract 84
4.1 Introduction 85
4.2 Materials and methods 86
4.2.1 Animals and feeding 86
4.2.2 Feed sampling and chemical analyses 88
4.2.3 Defaunation of lambs 89
4.2.4 Blood methaemoglobin 89
4.2.5 Methane production 90
4.2.6 Digestibility, digesta kinetics and microbial protein outflow 90
4.2.7 Rumen fluid sampling, ammonia, volatile fatty acid concentrations, and protozoal
enumeration 92
4.2.8 Liveweight and clean wool growth 92
4.2.9 Statistical analyses 93
4.3 Results 93
4.3.1 Blood methaemoglobin concentration 93
4.3.2 Rumen fermentation and methane emissions 95
4.3.3 Performances and digestion 96
4.4 Discussion 98
4.4.1 Effects of nitrate on blood MetHb concentration and protozoa population 98
4.4.2 Effects of nitrate supplementation and defaunation on performances and digestion 99
4.4.3 Effects of nitrate supplementation and defaunation on methane emissions and rumen
fermentation 101
4.4.4 Interaction of defaunation and nitrate supplementation 103
4.5 Conclusion 104
Chapter 5 107
Effects of rumen protozoa of Brahman heifers and nitrate on fermentation and in
vitro methane production 107
Abstract 108
5.1 Introduction 109
5.2 Materials and methods 110
5.2.1 Animals and feeding 110
viii
5.2.2 Defaunation of cattle 111
5.2.3 Refaunation of cattle 111
5.2.4 Rumen fluid sampling, ammonia, volatile fatty acid concentrations, and protozoal
enumeration 112
5.2.5 In vitro incubations and measurements 113
5.2.6 Statistical analyses 114
5.3 Results 114
5.3.1 Protozoal population in refaunated heifers 114
5.3.2 Fermentation pattern and methane production in Experiment 1 116
5.3.3 Fermentation pattern and methane production in Experiment 2 118
5.4 Discussion 119
5.5 Conclusion 122
Chapter 6 125
Effects of defaunation and dietary coconut oil distillate on fermentation, digesta
kinetics and methane production of Brahman heifers 125
Abstract 126
6.1 Introduction 127
6.2 Materials and methods 128
6.2.1 Animals and feeding 128
6.2.2 Feed sampling and chemical analyses 129
6.2.3 Defaunation of cattle 130
6.2.4 Refaunation of cattle 132
6.2.5 Rumen fluid sampling, ammonia, volatile fatty acid concentrations, and protozoal
enumeration 132
6.2.6 Methane production measurement 133
6.2.7 Digesta kinetics and estimation of microbial protein supply 134
6.2.8 Statistical analyses 135
6.3 Results 135
6.3.1 Protozoal populations 135
6.3.2 Rumen pH, volatile fatty acid and ammonia concentrations 136
6.3.3 Methane emissions 138
6.3.4 Dry matter intake, digestibility, digesta kinetics, microbial protein outflow and liveweight
change 139
6.4 Discussion 140
6.4.1 Protozoal population in refaunated heifers after inoculation and effect of coconut oil
distillate on protozoal population 141
6.4.2 Effects of defaunation treatment 142
6.4.3 Effects of coconut oil distillate supplementation 145
6.5 Conclusion 148
ix
Chapter 7 151
Distribution of ciliate protozoa populations in the rumen, reticulum, and omasum of
Angus heifers 151
Abstract 152
7.1 Introduction 152
7.2 Materials and methods 153
7.2.1 Animals, feed and sampling 153
7.2.2 Sample processing and protozoal enumeration 155
7.2.3 Statistical analyses 156
7.3 Results 157
7.4 Discussion 159
7.5 Conclusion 163
Chapter 8 167
General discussion 167
8.1 Introduction 167
8.2 Protozoal impacts on the rumen and its fermentation and methane production 168
8.3 Growth and productivity of defaunated ruminants 172
8.4 Defaunation and dietary oil or nitrate as complementary mitigation strategies 174
8.5 Challenges to applying defaunation in commercial practice 176
8.6 Conclusion 177
References 179
x
List of Figures
Chapter 1
Figure 1.1 Greenhouse gas sources from Australia’s agricultural sector 2
Figure 1.2 Pathways leading to VFA and methane production in glucose 3
Figure 1.3 The relationship between enteric methane production and liveweight gain of cattle. 5
Chapter 2
Figure 2.1 Protozoal population of the inoculum and of refaunated sheep on day 7, 14, 21, 30 and 38
after protozoa inoculation. 44
Figure 2.2 Relationship between volume and weigh of rumen-reticulum estimated by CT scan. 48
Chapter 3
Figure 3.1 The relationship between pasture green dry matter biomass (Green DM; kg DM/ha) and
normalised difference vegetation index (NDVI). 67
Chapter 5
Figure 5.1 Small Isotrichs (□), large Isotrichs (■) and small Entodiniomorphs (░) from refaunated
heifers 7, 14 and 21 days after refaunation. 115
Figure 5.2 Methane production (□) and protozoal numbers (■) in rumen fluid from refaunated heifers
0, 7, 14 and 21 days after refaunation using a mixed rumen fluid inoculum. 115
Chapter 6
Figure 6.1 Relationship between protozoa (-/+P) and COD supplement (-/+COD) on ruminal NH3-N
concentration. 137
Figure 6.2 Relationship between protozoa (-/+P) and COD supplement (-/+COD) on microbial crude
protein outflow. A common suffix above error bars indicate non-significant difference. 140
Chapter 7
Figure 7.1 Protozoal population densities (cells/mL) in rumen fluid from Angus heifers receiving
lucerne cereal hay mix over a period of 42 days. 158
xi
List of Tables
Chapter 1
Table 1.1 Characteristics of some rumen entodiniomorphid protozoa (Williams and Coleman 1988). 7
Table 1.2 Characteristics of some rumen holotrich ciliates (Williams and Coleman 1992). 8
Table 1.3 Characteristics of some rumen flagellate protozoa (Williams and Coleman 1992). 9
Chapter 2
Table 2.1 Experimental schedule for the defaunation, refaunation and data measurements. 36
Table 2.2 Chemical composition of the pellets supplied through the Greenfeed Emision Monitoring
(GEM) unit and of oaten chaff (g/100 g dry matter). 37
Table 2.3 Weekly rumen fermentation characteristics, intake and methane emission of defaunated (-P)
sheep and of refaunated (+P) sheep before (Day 0) and up to 21 days after protozoa inoculation. 45
Table 2.4 Reticulo-rumen (RR) volume, weight, gas volume and gas proportion of defaunated (- P)
sheep and of refaunated (+ P) sheep. Day 0 data was used as a covariate. 47
Table 2.5 Rumen fermentation characteristics, feed intake, methane emission and nutrient utilisation
of defaunated (- P) sheep and of refaunated (+ P) sheep offered a fixed intake. 49
Chapter 3
Table 3.1 Experimental schedule for pasture rotation and data measurements. 65
Table 3.2 Pasture green dry matter (GDM), pasture green fraction and chemical analysis of the
pastures‡ available to defaunated (-P) and faunated (+P) sheep rotationally grazing. 68
Table 3.3 Crude protein, dry matter digestibility and metabolisable energy content of pastures on offer
and that estimated to be selected by defaunated (-P) and faunated (+P) sheep using GrazFeed (Freer et
al. 1997). 69
Table 3.4 Chemical composition of the pellets supplied through the GreenFeed Emision Monitoring
(GEM) unit (g/100 g dry matter). 70
Table 3.5 Intake, rumen fermentation parameters and methane emissions of defaunated (-P) and
refaunated sheep (+P) grazing pastures. 75
Table 3.6 Wool parameters of defaunated (-P) and refaunated sheep (+P) grazing pastures. 76
Chapter 4
Table 4.1 Chemical composition of the oaten chaff and nitrate-supplemented chaff. 88
xii
Table 4.2 Rumen fermentation characteristics, concentration of methaemaglobin (MetHb) and
protozoal population of defaunated (-P) and faunated lambs (+P) fed diets of oaten chaff with or
without nitrate (NO3) supplementation. 94
Table 4.3 Intake, productivity, methane emissions and digesta kinetics of defaunated (-P) and
faunated lambs (+P) fed diets of low-protein oaten chaff with/without nitrate supplementation. 97
Chapter 5
Table 5.1 The pH, ammonia concentration and concentration and molar proportions of major volatile
fatty acids (VFA) in rumen fluid, and changes in gas and methane production in vitro after
refaunation. 117
Table 5.2 The pH, ammonia concentration, volatile fatty acid concentration and molar proportions
and methane production as influenced by the presence or absence of protozoa or nitrate addition in
incubations of rumen fluid in vitro. 118
Chapter 6
Table 6.1 Composition of the diets and fatty acid profile of coconut oil distillate. 130
Table 6.2 Experimental schedule for the defaunation, refaunation and data measurements. 127
Table 6.3 Enumeration of protozoa following refaunation in heifers and fed a diet containing either
4.5% coconut oil distillate (+COD) or nil (-COD). 136
Table 6.4 Physiological and rumen fermentation characteristics as influenced by the presence or
absence of protozoa or coconut oil distillate (COD) supplementation. 138
Chapter 7
Table 7.1 Chemical composition of the lucerne cereal hay mix (g/100g dry matter). 155
Table 7.2 Ciliate protozoa in reticular, ruminal and omasal contents and adhering to the gut tissues of
Angus heifers. 159
Chapter 8
Table 8.1 Rumen metabolite concentration and methane production in the rumen fluid of defaunated
animals normalized relative to those in faunated animals (1.00). Data are from experiments in this
thesis and from published reviews. 169
Table 8.2 Dry matter intake, digestibility, microbial protein outflow, liveweight gain and wool growth
of defaunated ruminants normalized relative to those of faunated ruminants (1.00). Data are from
experiments in this thesis and from published reviews. 173
xiii
List of abbreviations
ADF Acid detergent fibre
ADG Average daily gain
CH4 Methane
CO Coconut oil
CO2 Carbon dioxide
CP Crude protein
CWG Clean wool growth
DM Dry matter
DMD Dry matter digestibility
DMI Dry matter intake
DMP Daily methane production
FCR Feed conversion ratio
GEM GreenFeed emission monitor
GHG Greenhouse gases
H2 Hydrogen
LC Lucerne chaff
LW Liveweight
MCFA Medium-chain fatty acid
ME Metabolisable energy
MetHb Methaemoglobin
MI Methane intensity
MJ Mega joules
MP Methane production
MY Methane yield
N Nitrogen
NDF Neutral detergent fibre
NO3 Nitrate
NPN Non protein nitrogen
OC Oaten chaff
OM Organic matter
RR Reticulo-rumen
VFA Volatile fatty acid
Chapter 1: Literature review
1
Chapter 1
Review of the literature
1.1 Overview
Australia’s agricultural sector produces an estimated 87.4 Mt of greenhouse gas
emissions (GHG; expressed as CO2 equivalent; CO2-e) annually, contributing 16% of
national GHG emissions. Enteric fermentation is the main source of agricultural GHG
emissions accounting for 64.3% of Australia’s agricultural emissions (56.2 Mt CO2-e;
Figure 1.1). Livestock enteric fermentation is not only Australia’s largest agricultural
GHG emission source but it also contributes 11.6% of GHG from anthropogenic
sources globally (Ripple et al. 2014). In association with an increased global human
demand for food, agricultural demand for livestock products and consequently GHG
emissions are expected to increase in coming years (van Beek et al. 2010). Therefore,
mitigation strategies should focus on increasing animal efficiency and decreasing GHG
emissions per unit of edible food (Pinares-Patiño et al. 2009). Reduction of methane
emissions per unit of animal product will be achieved by means of reducing the
proportion of energy consumed by livestock that is expended in maintenance and
directing more towards faster growth, milk yield and shorter dry period in lactating
cows (Monteny et al. 2006).
Chapter 1: Literature review
4
1.1.2 The management of enteric methane
Enteric CH4 is a by-product of microbial fermentative digestion in the rumen of
ruminants that also represents a loss of 5 to 7% of gross energy intake, equivalent to a
CH4 yield of 16 to 26 g CH4/kg of dry matter (DM) consumed (Hristov et al. 2013). In
addition, animals, that grow faster, produce less CH4 per kg of ADG on the same
quality of diets (Figure 1.3). Theoretically, enteric CH4 emissions from ruminants can
be managed or mitigated by several potential strategies as summarised below.
Diet manipulation to reduce CH4 release per unit of animal production (Hristov et
al. 2013). This strategy is achievable by feeding animals highly digestible diets, but
it is less feasible in the many developing countries where residues of crops and
agricultural by-products are the major feed sources for livestock (Preston 1995).
Addition of feed additives that inhibit methanogenesis including
bromochloromethane (Hristov et al. 2013) and plant secondary compounds such as
saponins and tannins (Patra and Saxena 2009) or additives such as dietary fatty
acids that have a high affinity for bioreduction (Patra 2014). However, these
secondary compounds need to be well understood in regard to their sources,
absorption, metabolism and biological effects on livestock health and production
before they can be used in commercial livestock production systems (Durmic and
Blache 2012).
Removal of the rumen protozoa (defaunation). Methanogens, which exist as endo-
and ecto-symbionts with ciliate protozoa (Finlay et al. 1994; Tokura et al. 1997),
had been estimated to account for 37% of CH4 production (Finlay et al. 1994).
Chapter 1: Literature review
6
1.2 The rumen protozoa and classification
Protozoa were first discovered by Leeuwenhoek in 1675, and various protozoa were
identified as ciliates as their locomotion was made by the means of small hairs or cilia
over the surface of the body. Through evolution, as ruminants consumed grass and
water, the protozoa established in stomachs of ruminants and adapted to utilise this
habitat (Hungate 1966). Anaerobic rumen ciliates are extremely abundant, ranging from
105 to 10
6 cells/mL of rumen liquor and are capable of engulfing bacteria and digesting
plant materials such as cellulose and other structural carbohydrates (Finlay and Esteban
2013; Esteban et al. 2014).
Rumen protozoa are classified based on their cell morphology, and considered to be the
simplest form of animal life, performing all the life processes as a eukaryotic cell
(Dehority 2003; Esteban et al. 2014). Further, the majority of protozoa identified in the
rumen are ciliate species, with more than 100 species of rumen ciliate protozoa having
been identified in two major sub-classes; being the entodiniomorphid (Table 1.1) and
the holotrich ciliates (Table 1.2; Williams and Coleman 1988, 1992). A few species of
flagellate protozoa (Table 1.3) are also found in the rumen (Hungate 1966; Williams
and Coleman 1992). However, the flagellates are easily confused with fungal zoospores
(Dehority 2003). In addition, flagellate protozoa are less numerous in terms of
population density and have small body mass compared to the ciliates (Hungate 1966;
Clarke 1977). Therefore, the flagellates are not well known and have not been the focus
of attention in classifying or describing their activity and metabolism, leading to little
information being available.
Chapter 1: Literature review
7
Table 1.1 Characteristics of some rumen entodiniomorphid protozoa (Williams and
Coleman 1988).
Genus Dorsal cilia Obvious
skeletal plates
Macronucleus
shape
Length
(µm)
Width
(µm)
Entodinium 0 0 Various 22-29 11-68
Eodinium 1 band ant.end‡
0 Rod-shaped 32-60 20-40
Diplodinium 1 band ant.end 0 Often bent rod 55-210 41-136
Eremoplastron 1 band ant.end 1 narrow Often bent rod 45-500 21-260
Eudiplodinium 1 band ant.end 1 narrow Hook shaped 105-198 56-120
Ostracodium 1 band ant.end 1 wide Various 58-133 36-54
Polyplastron 1 band ant.end 2 narrow Rod-shaped 123-205 98-123
Diploplastron 1 band ant.end 2 narrow close at
post. End Rod-shaped 88-120 47-65
Metadinium 1 band ant.end 2 narrow occ.
Fused
Rod-shaped 2-
3 lobes 110-288 61-165
Epidinium 1 band behind
ant.end 3 variable width Elongate 105-150 44-72
Enoploplastron 1 band ant.end 3 narrow close
together Elongate 60-140 32-90
Ophryoscolex 1 band round
3/4 of middle 3 variable width Elongate 120-215 60-80
Epiplastron 1 band round
3/4 of middle 5 variables width Elongate 90-140 41-60
Elytroplastron 1 band ant.end 3 narrow (2 on
right 1 on left) Elongate 110-160 67-97
Caloscolex 1 band round
all middle 1 complex Elongate 130-160 73-90
Opisthotrichum 1 band round
1/3 of middle 1 cylindrical Elongate 60-80 21-28
Parentodium 0 0 Round 26-39 14-21
‡ anterior end
Chapter 1: Literature review
8
Table 1.2 Characteristics of some rumen holotrich ciliates (Williams and Coleman
1992).
Species Morphology Size range,
average (µm)
Length: width
range, average Macronucleus
Isotricha prostoma
Elongated
ovoid to
elipsoidal
80-200×50-
120; 135×70
1.69-2.55;
2.03 Elongated
Isotricha
intestinalis
Elongated
ovoid to
elipsoidal
90-200×45-
150; 110×60
1.65-1.93;
1.76 Ovoid, 30×20
Dasytricha
ruminantium Ovoid
35-75×20-40;
57×27
1.70-2.70;
2.11
Elongated/ellipsoidal,
16-18×8-9
Dasytricha
hukuokaensis Ovoid
120-180×68-
122; 151×95
1.47-1.76;
1.59
Ellipsoildal, 24-
38×16-20; 31×18
Oligoisotricha
bubali Ovoid
12-20×8-15;
16×12
1.07-1.60;
1.30 Spherical-elliptical
Buetschlia parva Ovoid 30-67×20-48;
55×35
1.58-2.38;
1.91 Spherical
Buetschlia
neglecta Ovoid 40-60×20-30 2.0 Spherical
Buetschlia
lanceolate Spear-shaded 48×20 2.4 Large
Buetschlia
omnivore Ovoid/spherical
Variable; 35-
110×27-97 Elongated
Buetschlia nana Ovoid 17-21×12-
17;19×15 spherical
Parabundleia
ruminantium Ovoid
37.5-50 ×
27.5-32.5;
42.5 × 30.5
1.25-1.54 Elliptical, 16 µm
long
Polymorphella
bovis
Ovoid to bottle-
like
26-37.5 × 20-
26; 34× 22
1.30-1.80
1.56
Subspherical, 2.5 µm
long
Blepharoprosthium
parvum Pyriform
26-32 × 16-
20; 29 × 18 Spherical
Blepharoconus
krugerensis
Ovoid with
anterior
knoblike
protuberence
30-65 × 21-
60; 46 × 35
1.11-1.80;
1.34
Disc-shaped 7-15 ×
4-8; 11 × 5.5 µm
Microcetus lappus Ovoid/elongate 18-29 × 7.5-
18; 23.6 × 13 Spherical
Chapter 1: Literature review
9
Table 1.3 Characteristics of some rumen flagellate protozoa (Williams and Coleman
1992).
Species Shape Size Number of
flagella
Size of nuclear
(µm)
Chilomastix caprae Piriform 8.3×4.4 4
Monocercomonas
ruminantium Piriform 4.8×4.1 4 1.8×1.6
Monocercomonoide
bovis Elliptical 5.4×2.8 4 1.6×1.4
Monocercomonoide
caprae Elliptical 9×6 4 Large
Pentatrichomonas
hominis Elliptical 7.5×5.6 5 2.5×2.0
Tetratrichomonas
buttreyi Elliptical 5.3×4.8 4 2.0×1.7
Trichomonas
ruminantium Elliptical 12×10 3
1.3 Role of protozoa in ruminant nutrition
Rumen protozoa account for as much as half the total microbial biomass in the rumen
and up to 60% of total fermentation products (Williams and Coleman 1992; Newbold et
al. 2015). They actively participate in the ruminant digestion process. Removing
protozoa from the rumen, therefore, may result in modifying ruminal digestion of plant
cell walls and starch which are considered to be two main sources of energy supply for
ruminants (Jouany and Martin 1997). The ruminal ecosystem and environment can be
slightly altered by the absence of protozoa with significant influence on bacterial
activity, affecting the retention time of the digesta, the concentrations and proportion of
ruminal VFA and ammonia (NH3) concentration (Eugène et al. 2004a; Newbold et al.
2015) and therefore the supply of metabolites to the host, especially amino acids.
Chapter 1: Literature review
10
1.3.1 Plant cell wall digestion
Early studies on the role of rumen protozoa in ruminal digestion concluded that rumen
protozoa did not digest plant cell components (Becker 1929). In later years, enzymatic
and microscopic evidence showed that cellulose was digested by entodiniomorphs
(Hungate 1966). Further, the rumen ciliate protozoa were confirmed in their ability to
colonize and damage plant tissues in studies with scanning electron microscopic
technology. Epidinium crawley was found to cause primary degradation of plant tissues
(Bauchop and Clarke 1976) as it attached and damaged areas of the stem. Entodinium
spp. and Ophryoscolex caudatum, however, rarely attached themselves to the large
tissue fragments, but to damaged tissues exposed through fractures (Orpin 1984).
Rumen protozoa produce fibre-degrading enzymes and the cellulolytic enzymes
produced by rumen protozoa are distinguished from those of bacterial and fungal origin.
This was observed by the characterization of protozoal genes encoding cellulase
enzymes (Jouany and Martin 1997). Further, polysaccharidase activities were greater in
animals with ciliate protozoa compared to animals without ciliate protozoa (Santra and
Karim 2002; Eugène et al. 2004b). However, effects of rumen protozoa on ruminal
digestion are inconsistent in the literature. There are reports of decreased organic matter
(OM) digestibility (Eugène et al. 2004a; Newbold et al. 2015), and neutral-detergent
fibre (NDF) and acid-detergent fibre (ADF) digestibility (Newbold et al. 2015) in the
absence of rumen protozoa, probably due to the loss of protozoal fibrolytic activity. The
absence of rumen protozoa, in contrast, increases cellulolytic ruminococci populations
(Mosoni et al. 2011). This increased cellulolytic ruminococci in the absence of rumen
Chapter 1: Literature review
11
protozoa may compensate for the loss of protozoal fibrolytic activity, and therefore the
digestibility of OM, NDF and ADF was not different between defaunated and faunated
animals (Jouany et al. 1995). Zeitz et al. (2012) observed no effects of individual ciliate
protozoa such as Entodinium caudatum, Epidinium ecaudatum and Endiplodium maggii
on whole tract digestibility of OM, NDF or ADF and suggested that ciliate protozoa
may not always improve plant cell wall digestion.
1.3.2 Carbohydrate and starch digestion
Both holotrich and entodiniomorph ciliate protozoa are believed to ferment
carbohydrates to meet their energy requirement. Holotrich ciliates utilise soluble
carbohydrates, while Entodinium spp. and Epidinium spp. preferably digest starch
(Williams 1989). De Smet et al. (1992) showed that the total protozoal population
density was nearly double in a high concentrate diet compared to a high roughage diet,
with holotrich ciliates increasing accordingly. Entodinium spp. and Epidinium spp.
have their largest numbers with a high concentrate diet.
Wereszka and Michałowski (2012) found that Diploplastron affine possessed enzymes
degrading starch and its derivatives. A protozoal cell extract for enzymatic studies
found Diploplastron affine ciliate capable of digesting starch, released about 45 pmol
VFA per protozoa per hour and utilised liberated energy for their energy requirement.
The rate of starch degraded by Diploplastron affine is equivalent to 2.4 ± 0.47μmol/L
glucose per mg protein per min and the degradation rate of maltose is approximately
0.05μmol/L glucose per mg protein per min (Wereszka and Michałowski 2012). The
ciliate Diploplastron affine is also found to digest insoluble 1,3-ß-glucans such as
Chapter 1: Literature review
12
pachyman and 1,6-ß-glucans such as pustulan as energy substrates (Belzecki et al.
2012).
Apart from being able to digest and utilise plant carbohydrates, rumen protozoa also
ferment chitin of the rumen fungus. Morgavi et al. (1994) found Piromyces spp. strain
OTS1 in monocultures or in the presence of rumen protozoa in vitro and reported that
rumen protozoa adversely affect the growth of Piromyces spp. strain OTS1 and are able
to digest fungal cell walls, resulting in 42% reduction of chitin which is a carbohydrate
component of the fungal cell wall. Diploplastron affine and Entodinium caudatum are
found to possess a chitinolytic enzyme (Miltko et al. 2015b) and utilise chitin as a
source of energy for ciliate metabolism (Miltko et al. 2015a).
1.3.3 Protein digestion and protozoal synthesis in the rumen
Ruminants with protozoa in the rumen (faunated) support a higher ruminal NH3
concentration than do animals with rumen protozoa removed (defaunated), indicating
that rumen protozoa degrade dietary proteins (Jouany 1996) and engulf bacteria for their
amino acid requirement (Coleman 1989; Esteban et al. 2014). Ueda et al. (1975) who
incubated the isotrich ciliates with soluble casein found that peptide-nitrogen and
amino-nitrogen produced by the isotrich reached its highest level between 3 and 15
hours of incubation, accounting for 47% and 58% of non-protein nitrogen (NPN),
respectively. Incubation of the ophryoscolecid ciliates with insoluble casein showed a
peak of peptide-nitrogen at 3 hours, which accounted for 36% of NPN while amino-
nitrogen increased linearly and accounted for 47% of NPN at 15 hours of incubation
(Ueda et al. 1975).
Chapter 1: Literature review
13
Protozoal nitrogen (N) can account for 53% of the total microbial N in the bovine
rumen (Michałowski 1979), which is about 24 to 46 g N (Leng et al. 1981). However,
rumen protozoa contribute only 20% of the total microbial N entering the duodenum
(Jouany et al. 1988). The smaller protozoal biomass in the duodenum of the ruminants
could be due to 65% to 74% of protozoa lysing and being degraded in the rumen (Leng
1982; Ffoulkes and Leng 1988), suggesting only 24 to 35% of protozoa enter the lower
digestive tract. The relatively high numbers of rumen protozoa that complete their life
span in the rumen (Leng 1982) and are retained within the omasum of ruminants
(Czerkawski 1987) mean rumen protozoa contribute a small proportion of the total
microbial protein supply. The principal detrimental effect of rumen protozoa, therefore,
may be competition for substrate with bacteria and engulfment and digestion of bacteria
by protozoa, leading to decreased bacterial biomass and flow of protein in the
duodenum (Leng 1982).
1.3.4 Ruminal lipid metabolism
The role of rumen protozoa in bio-hydrogenation is not well defined and understood
(Williams and Coleman 1992), although they contribute significantly to flow of
unsaturated fatty acids to the duodenum (Newbold et al. 2015; Yáñez-Ruiz et al. 2006).
Yáñez-Ruiz et al. (2006) reported rumen protozoa accounted for between 30-40% of
conjugated linoleic acid (CLA) and 40% of vaccenic acid (VA) leaving the rumen.
Mixed protozoa from the sheep rumen contain at least two to three times more
unsaturated fatty acids, including CLA and VA, than do bacteria. Different species have
different composition, with larger fibrolytic species such as Epidinium ecaudatum
Chapter 1: Literature review
14
caudatum containing more than ten times more CLA and VA than some small species,
including Entodinium nanellum (Devillard et al. 2009). This high level of
polyunsaturated fatty acids in protozoal cells is a consequence of ingestion and/or
engulfment of chloroplasts (Huws et al. 2009) and this chloroplasts uptake is specifically
found in entodiniomorphids (Huws et al. 2012). Rumen protozoa, therefore appear to
increase the duodenal flow of mono or polyunsaturated fatty acids by protecting
chloroplasts unsaturated fatty acids from rumen bio-hydrogenation.
1.4 Factors affecting protozoal population densities in the
rumen
Rumen ciliate protozoa represent approximately 104-10
6 cells/mL of rumen contents
(Dehority 2003; Esteban et al. 2014), but the concentration of rumen protozoa varies
among animals and is dependent on many factors such as ruminant species,
geographical location (Akbar et al. 2009), diet (Whitelaw et al. 1984), frequency of
feeding and rumen pH (Clarke 1977).
1.4.1 Diet composition
Ruminants fed highly digestible diets often show the largest populations of rumen
protozoa (Hungate 1966), while small populations of rumen protozoa are found in
animals on low quality roughage diets (Abe et al. 1973). De Smet et al. (1992) fed
sheep low and high concentrate diets containing 4.3% or 17.3% starch respectively,
observing total protozoal population was nearly two-fold higher in the high concentrate
diet. Rumen protozoa are able to reduce the rate of fermentation, contributing to the
maintenance of a stable ruminal ecosystem when a high concentration of grain is
Chapter 1: Literature review
15
suddenly introduced in the diet (Mackie et al. 1978). However, the protozoa are
significantly affected by the environment’s acidity or alkalinity, with the protozoa
unable to survive if rumen pH is above 7.8 or below 5.0 (Clarke 1977). Mackie et al.
(1978) also found that the protozoal population decreased by 50-80% as rumen pH fell
below 5.4. Cellulolytic ciliates almost disappeared when cattle were fed barley only
(Kudo et al. 1990) and steers became protozoa-free for a period of a few weeks by ad
libitum feeding of barley (Whitelaw et al. 1984).
1.4.2 Dietary fatty acid supplement
Capric acid (C10:0), lauric acid (C12:0) and myristic acid (C14:0) show strong
protozoal toxicity and are useful rumen defaunating agents (Matsumoto et al. 1991).
Matsumoto et al. (1991) observed that rumen protozoa, except Entodinium spp. were
undetectable after 3 days of feeding 30 g of hydrated coconut oil (CO) containing 52%
lauric acid. Feeding 250g of refined CO to beef heifers reduced rumen protozoal
population by 62% (Jordan et al. 2006) and protozoal populations in beef heifers were
decreased by 63% and 80% by 300 g/d CO after 45 and 75 days, respectively (Lovett et
al. 2003). Machmüller (2006) reported a reduction in rumen protozoa by 88 and 97%
when feeding sheep with 3.5 and 7% CO, respectively. This suppressive effect of CO on
rumen protozoa persisted 5 weeks after finishing feeding sheep with CO (Sutton et al.
1983). Rumen protozoa were reduced to half of the original population by cottonseed,
with holotrich and cellulolytic protozoa apparently lost from the rumen of sheep and
only Entodinium spp. remained (Dayani et al. 2007).
Chapter 1: Literature review
16
1.4.3 Frequency of feeding
The concentration of protozoa in the rumen liquor varies according to the daily feeding
regime, reaching a maximum before feeding and decreasing by approximately 60-80%
from 4 to 12 hours after feeding (Michalowski and Muszyński 1978). More specifically,
the holotrich population in the fluid decreases for a period of 12 to 20 hours after
feeding and the population returns to its original numbers within 4 to 6 hours pre-
feeding, while the entodiniomorphid population in the fluid decreases for up to 16 hours
after feeding and then increases to the pre-feeding numbers (Williams 1986). The
increase of the holotrich population is mainly caused by the increase in dasytrichs while
the isotrich population remains relatively low (Clarke 1965). The highest concentration
of rumen protozoa occurs when the animal is fed three or four meals per day rather than
once (Bonhomme 1990).
1.5 Defaunating the rumen
Rumen protozoa are important, but not essential in the rumen ecosystem and to the
well-being of host animals (Williams and Coleman 1992; Newbold et al. 2015).
Elimination of rumen protozoa (defaunation) has led to reported increases in growth rate
and liveweight gain of ruminants (Bird and Leng 1978; Bird et al. 1979; Eugène et al.
2004a; Newbold et al. 2015) especially when the feed is deficient in protein relative to
energy content. In addition, rumen protozoa are significant hydrogen (H2) producers and
synthesise mainly acetate and butyrate rather than propionate (Williams and Coleman
1992). Defaunation is therefore expected to induce a greater proportion of propionate in
the ruminal VFA (Eugène et al. 2004a), but this phenomenon is not always observed
Chapter 1: Literature review
17
(Williams and Coleman 1992; Newbold et al. 2015). The reduced CH4 emissions
caused by defaunation also reported by several authors (Whitelaw et al. 1984; Hegarty
1999; McAllister and Newbold 2008; Newbold et al. 2015) may reflect reduced H2
availability by removing endosymbiotic methanogens (Finlay et al. 1994; Tokura et al.
1997; Finlay and Esteban 2013). To attain desired fermentation and productivity
advantages from defaunation, many defaunation strategies have been tried and are
summarised below.
1.5.1 Isolation of young ruminants after birth
Rumen ciliates are not observed in new born animals, but that rumen ciliates are passed
from mother to baby by direct transfer of saliva containing the active protozoa (Stewart
et al. 1988). Fonty et al. (1986) found protozoa appeared in lambs in the following
order: Entodinium (15-20 days), Polyplastron, Eudiplodinium (20-25 days) and
Isotricha (50 days).
Therefore, rumen ciliate protozoa are not present in animals at birth, enabling protozoa-
free animals to be established by separating offspring from their mothers (Ivan et al.
1986). Bryant and Small (1960) reared calves isolated from birth which did not have
ciliate protozoa until they were inoculated with rumen contents at 24 weeks of age.
Eadie and Gill (1971) separated lambs from their dams at 2 days of age and maintained
protozoa-free lambs for 61 weeks during the length of the experiment. Dehority (1978)
also isolated lambs for almost a year without protozoa until the sheep were inoculated
with rumen contents to faunate them. In addition, Hegarty et al. (2008) established a
flock of ciliate-free lambs born from defaunated ewes and the lambs remained protozoa-
Chapter 1: Literature review
18
free for an extended period of time while grazing. It seems probable young ruminants
can be reared free of protozoa when isolated after birth, but it is time consuming and it
is not applicable to adult ruminants.
1.5.2 Chemical drenching methods
Chemicals administered into the rumen by using an oesophageal tube or through a
rumen fistula have been shown to eliminate the rumen protozoa. A chemical dosing
method was described by Becker (1929) who fasted goats for three days and at the end
of 72 hours, goats were dosed with 50 mL of 2% copper sulphate for two consecutive
days. Jouany et al. (1988) repeated this method and reported 50% of treated sheep died
of copper poisoning and only one sheep was protozoa-free for 93 days. This protocol
was therefore considered dangerous and unreliable.
Rumen ciliate protozoa are susceptible to surface-active agents and these agents provide
an effective protocol for defaunation of the rumen. In sheep, defaunation has been
successful with sodium 1-(2-sulfonatooxyethoxy) dodecane (Bird et al. 2008; Hegarty
et al. 2008) or sodium lauryl sulfate (Santra et al. 2007a). In cattle, removing protozoa
with chemical treatment appears more challenging. Diocyl sodium sulfosuccinate
(Manoxol OT) used by Abou Akkada et al. (1968), and non-ionic surfactants such as
nonyl-phenol ethoxylate (Teric GN9) used by Bird and Leng (1978) were not successful
in rendering cattle free of ciliate protozoa for prolonged periods. These surface-active
agents are more effective to use in sheep than in cattle, but even defaunating sheep with
surfactants is not always successful (Machmüller et al. 2003).
Chapter 1: Literature review
19
Part of the difficulty in chemical defaunation may be due to protozoa residing in the
omasum. The omasum transfers digesta from reticulum into the abomasum and has a
major role in water reabsorption (Van Soest 1994). The flow of digesta from the
reticulum occurs following the omasal canal contractions, but occasionally backflow of
large volumes of digesta from the omasum to the reticulum occurs when the omasal
body contracts during the closure of omaso-abomasal orifice (Stevens et al. 1960).
Therefore, Towne and Nagaraja (1990) claimed that the backflow of omasal contents
containing residual omasal protozoa would re-inoculate the rumen of the defaunated
rumen in steers. Towne and Nagaraja (1990) also suggested that anatomical differences
between bovine and ovine omasums affect the efficacy of chemical defaunation because
the number of omasal laminae in cattle are almost double those in sheep. Chemicals are
therefore less likely to reach all protozoa residing between laminae in bovines than in
sheep, and this may explain why reports of successful defaunation in cattle are rare.
1.5.3 Effect of defaunation on extent of ruminal fermentation
An in vitro study by Yoder et al. (1966) reported cellulose digestion by rumen protozoa
(7%), by bacteria (40%) and by protozoa and bacteria combined (exceeded 60%),
showing a beneficial effect of rumen protozoa on cellulose digestion. Bauchop and
Clarke (1976) observed rumen ciliate protozoa contribute to fibre digestion as they are
capable of colonizing and damaging plant tissues. Cellulolytic, polysaccharide
depolymerases and glycoside hydrolase enzymes produced by protozoa are significant
contributors to cellulose and hemicellulose fermentation (Coleman 1989). Therefore,
Chapter 1: Literature review
20
the absence of rumen protozoa can lead to a 5-15% reduction in carbohydrate digestion
of plant cell walls (Jouany et al. 1988).
Removing protozoa reduces the rumen digestibility of fibre components of the diet
(Newbold et al. 2015). Ruminal digestion of NDF and ADF were reduced by 31% and
22% respectively by defaunation of sheep when fed a low soluble N diet (Ushida and
Jouany 1990). Defaunation also reduced degradation of a mainly chopped hay diet by
up to 18% in sacco (De Smet et al. 1992). The absence of rumen protozoa did not affect
rumen digestibility in lambs offered a diet with a high protein/energy ratio, but reduced
total tract digestibility of OM (10%) and NDF (7%; Eugène et al. 2010). In addition,
Ushida and Jouany (1990) found that defaunated ruminants require an increased supply
of NPN in order to maintain fibrolytic activity in the rumen compared to faunated
animals. Although fibre digestion is moderately suppressed by defaunation, improving
protein supply is far more important in growing animals with high protein demand and
when protein is a limiting factor in the diet (De Smet et al. 1992).
1.5.4 Effect of defaunation on ruminal volatile fatty acids
The effects of defaunation on ruminal VFA concentration and the molar proportions of
VFA are not entirely consistent within the literature (Williams and Coleman 1992;
Newbold et al. 2015). Defaunation has been reported to increase total VFA
concentration in defaunated sheep (Santra et al. 2007a) and weaner lambs (Santra and
Karim 2002), but Hegarty et al. (2008) reported that animals with protozoa had higher
concentrations of total VFA compared with defaunated animals. Molar proportions of
VFA are also inconsistently affected by defaunation, although butyrate and acetate
Chapter 1: Literature review
21
proportions were generally increased (Machmüller et al. 2003; Bird et al. 2008) and
proportion of propionate generally decreased after defaunation (Machmüller et al. 2003;
Hegarty et al. 2008). A higher proportion of acetate and lower proportion of propionate
in the VFA of defaunated animals was reported when animals were fed low-quality
diets (Bird 1982). In addition, Newbold et al. (2015) reported in their meta-analysis that
defaunation significantly decreased butyrate and increased acetate, but did not affect
propionate. These inconsistent effects of defaunation on VFA concentration and molar
proportions may reflect variable effects of defaunation on the bacterial population
within the rumen. Defaunation increases the number of bacteria, which induces changes
in digestion and fermentation due to bacterial species distribution (Mosoni et al. 2011;
Zeitz et al. 2012) and bacterial composition changes (Ozutsumi et al. 2005).
Elimination of ciliate protozoa from the rumen may allow a proliferation of rumen
bacteria, leading to increased uptake of NH3 by bacteria for protein synthesis with less
protein degraded by rumen protozoa (Williams and Coleman 1992; Jouany and Ushida
1999; Santra et al. 2007a). Decreases in NH3 concentration in defaunated animals
compared to faunated animals were observed in many studies (Eugène et al. 2004a;
Newbold et al. 2015). Less ruminal catabolism of engulfed feed-protein and bacteria
occurs in the absence of rumen protozoa, leading to an increase in the supply of protein
to the duodenum of ruminants for productive purposes (Bird and Leng 1978; Jouany
1996; Newbold et al. 2015).
Chapter 1: Literature review
22
1.5.5 Effect of defaunation on animal performance
A positive effect of defaunation on ruminants is the increased rumen bacterial biomass
and passage of ruminal undegraded protein from the diet (Jouany 1996). Duodenal N
and duodenal CP/kg DMI outflow significantly increased after defaunation (Eugène et
al. 2004a; Newbold et al. 2015), indicating an increase in the efficiency of microbial
protein synthesis and leading to an average increased daily gain of 11%.
In pen-feeding studies, defaunated lambs showed 18% faster growth rate and greater
wool growth and wool fibre diameter over faunated or refaunated lambs offered a 50:50
concentrate and roughage ration (Santra et al. 2007b). Birth weight of lambs born from
defaunated ewes was 13% heavier than from faunated ewes on single-born lamb and
pre-weaning growth rates were 10% and 14% heavier in lambs reared free of ciliate
protozoa for both single and twin-born lambs, respectively (Hegarty et al. 2008). On
high energy and low protein diets, defaunated cattle grew at a 43% faster rate than
faunated cattle on the same intake (Bird and Leng 1978) and lambs without rumen
protozoa showed significantly increased growth rates and efficiency of utilisation of
feed when fed a low level of protein. Wool growth increased by 50% compared to
faunated animals that were fed a low protein diet (Bird et al. 1979).
In grazing studies, Bird and Leng (1984) observed a greater rate of body weight gain
(23%) and wool growth (19%) in defaunated compared to faunated lambs grazed on a
green oats pasture. Protozoa-free lambs born from defaunated ewes were significantly
(4-8%) heavier than were lambs born from faunated ewes measured from 2 months of
Chapter 1: Literature review
23
age to 5 months of age and wool growth was also greater in protozoa-free lambs grazed
on fescue dominant pastures (Hegarty et al. 2000).
However, defaunation has been associated with a 30% increase in rumen volume
(Orpin and Letcher 1984) and the increased weight of ruminal contents after
defaunation was probably due to longer particle retention of ruminal digesta associated
with the rumen fill effect of lower OM digestibility (Eugène et al. 2004a). There is little
data on carcass and gut weights of defaunated over faunated animals reported in the
literature.
1.5.6 Effect of defaunation on enteric methane production
As stated earlier, ciliate protozoa are significant producers of H2 and produce acetic and
butyric acids rather than propionic acid (Williams and Coleman 1992). Defaunation is
generally associated with fermentation shifting to a greater proportion of propionic acid,
therefore reducing the amount of CH4 produced (Eugène et al. 2004a).
The methanogens existing as endo- and ecto-symbionts with ciliate protozoa (Finlay et
al. 1994; Tokura et al. 1997; Finlay and Esteban 2013) have been estimated to account
for 37% of ruminal methane production (Finlay et al. 1994). The proportion of
methanogens in the total bacterial population was lower in protozoa-free lambs, with
26% lower CH4 emissions compared to faunated lambs (McAllister and Newbold
2008). While the archaeal community of methanogens in liquid and solid rumen
contents were similar in faunated wethers, a lower proportion of methanogens occurred
in the liquid phase with defaunation (Morgavi et al. 2012). However, Mosoni et al.
Chapter 1: Literature review
24
(2011) while observing a 20% reduction in CH4 emissions in short-term (10 week) and
long-term (2 year) defaunated sheep, found methanogens per gram of DM of rumen
content increased with defaunation while the diversity of the dominant methanogenic
community was not changed. Therefore, it may not be reasonable to attribute the
reduced CH4 production from defaunation to a loss of methanogens (Morgavi et al.
2012).
The presence of protozoa did not change enteric CH4 production in lambs raised
with/without protozoa from birth (Hegarty et al. 2008) or from 10 to 25 weeks after
chemical defaunation (Bird et al. 2008). Defaunation was associated with a reduced
number of methanogens in rumen fluid, but did not reduce CH4 production (Morgavi et
al. 2012; Kumar et al. 2013). This could be explained as defaunation induces changes in
bacterial or fungal populations (Eugène et al. 2004a) and the absence of protozoa in the
rumen leads to changes in the methanogen community (Morgavi et al. 2012).
Ruminal acetogens were found able to grow on CO2 and H2, and produce acetate, but
reductive acetogenesis was not likely to be occurring because of lower H2 affinity,
making reductive acetogenesis unable to compete with methanogens in the rumen
(Joblin 1999). In normal fermentation, methanogens reduce H2 to a low level in which
reductive acetogenesis is below detectable levels (Ungerfeld 2015), but if pyruvate-
derived acetate is produced when methanogenesis is inhibited, H2 may accumulate and
stimulate reductive acetogenesis (Ungerfeld 2013). Reductive acetogens established in
the rumen lacking methanogens can replace methanogens as a sink for H2 in the rumen
(Fonty et al. 2007). The reduced CH4 emissions from defaunated animals associated
Chapter 1: Literature review
25
with a rise in acetate proportion is a desirable condition in the rumen where reductive
acetogens may be occurring.
1.6 Effects of oils or nitrate on rumen fermentation and
methane emissions
A variety of strategies to manipulate rumen ecology to reduce enteric CH4 emissions
have been proposed. An immunization approach to control three selected methanogens
by vaccination achieved almost 8% reduced CH4 production in sheep (Wright et al.
2004). However, the diverse methanogenic community and the growth of untargeted
methanogens may account for the immunization failures (Williams et al. 2009). The use
of reductive acetogenesis is a potential approach to reduce CH4 production as acetogens
use H2 to reduce CO2 to acetate while methanogens reduce CO2 to CH4 (Ungerfeld
2013). In the rumen environment acetogens are less numerous and less efficient than
methanogens in the competition for H2 (Joblin 1999), thus results are often
unsatisfactory or not conclusive. The use of plant extracts to reduce CH4 is an
increasing interest as a natural alternative, but the use of plant extracts like tannin and
saponin has few studies tested on in vivo and results are highly variable (Martin et al.
2010). The use of essential oils, rich in medium chain fatty acids (MCFA), has shown a
promising dietary strategy to reduce CH4 emissions (Hristov et al. 2009; Patra 2014).
The reduced CH4 production by fatty acids is a result of reduced rumen protozoa and
methanogens (Matsumoto et al. 1991; Dohme et al. 2000; Liu et al. 2011). Besides
inhibitory effects of MCFA on rumen protozoa and methanogensis, addition of oils is
also used to increase the dietary energy (Coppock and Wilks 1991). However,
palatability of MCFA could reduce animal intake (Lovett et al. 2003; Hollmann et al.
Chapter 1: Literature review
26
2012) and reduce digestibility of DM and NDF, resulting in a disadvantage of increased
oils in diets when targeting a large amount of reduced CH4 emissions (Patra 2014).
Addition of nitrate (NO3) in diets has shown a persistent effect of reduced CH4
emissions from ruminants (van Zijderveld et al. 2011; Lee and Beauchemin 2014).
Stochiometrically, 1 mol of NO3 would produce 1 mol of ammonia and should reduce 1
mol or 16g of CH4 production. However, the potential toxicity of NO3 in the feed
associated with nitrite (NO2) poisoning constrains its use in practice (Leng and Preston
2010). A sufficient inclusion of NO3 in diets and a delivery practice is important to
reduce CH4 emissions with no effect on microbial fermentation because NO2 poisoning
occurs only when large quantities of NO3 in the feed suddenly introduces into rumen
without adaptation (Leng and Preston 2010; Lee and Beauchemin 2014). Nitrate is also
a major N source and it can be used to replace urea when ruminants are supported on
low protein forages (Nolan et al. 2010; Li et al. 2012).
1.6.1 Oils
The use of oils to kill methanogens and ciliate protozoa in the rumen and reduce CH4
production has been well reported. Methane production was reduced by 28% and 73%
in sheep fed with 3.5% and 7% of coconut oil (CO), but this did not significantly affect
energy metabolism or N turnover (Machmüller and Kreuzer 1999). Feeding CO
(50g/kg) significantly reduced CH4 emissions without affecting the total tract digestion
or energy retention of the sheep (Machmüller et al. 2003). Jordan et al. (2006) also
showed that feeding 250g/d of refined CO to beef heifers decreased CH4 output by 18%,
but maintained intake and improved animal performance.
Chapter 1: Literature review
27
A driver of decreased CH4 output by fatty acids may be the depression in DMI
following the CO supplementation (Sutton et al. 1983; Machmüller and Kreuzer 1999;
Lovett et al. 2003; Hollmann et al. 2012). This reduction in DMI was probably due to a
high level of lauric acid (42%) and myristic acid (15%) in CO. Hristov et al. (2011) and
Dohme et al. (2001) reported that lauric and myristic acid depressed DMI, ruminal fibre
degradation and had an adverse effect on ruminal fermentation. Nevertheless, a meta-
analysis of effects of dietary fat including sunflower oil, CO, linseed oil and corn oil by
Patra (2014) concluded that while ruminal NH3 concentration and digestibility of DM
and NDF were reduced linearly with increasing dietary fat, but total VFA and acetate
were not affected. The negative effects of dietary fat on digestibility of DM and NDF
were probably accounted for by the decrease in rumen protozoa and fibrolytic bacteria
(Patra 2014).
1.6.2 Dietary nitrate
Dietary NO3 has been shown to reduce CH4 emissions from ruminants with a consistent
and persistent efficacy (Nolan et al. 2010; van Zijderveld et al. 2010; van Zijderveld et
al. 2011; de Raphélis-Soissan et al. 2014); with a CH4 reduction range of 23% - 35%
when 1.9% to 2.6% NO3 was supplemented. A review by Leng and Preston (2010)
concluded that the use of NO3 as a H2 sink could reduce CH4 production from 16-50%,
depending on diets and the inclusion rate of NO3. This is because approximately 2
moles of H2 will be needed to convert NO3 to NO2 and 6 moles of H2 will be removed
in order to reduce NO2 to NH3 (Allison and Reddy 1984). Nitrate causes changes in
rumen fermentation, increasing acetate and decreasing propionate proportions as the
Chapter 1: Literature review
28
high affinity of NO3 for H2 is more favourable for NO2 formation than propionate or
CH4 formation (Ungerfeld and Kohn 2006). This reduces methanogenesis by
diminishing H2 availability (van Zijderveld et al. 2010; van Zijderveld et al. 2011).
Further, shorter mean fluid retention time in the rumen by supplementation of NO3 is
associated with a lower CH4 yield (Nolan et al. 2010). Also, NO3 can be potentially
used to replace urea as a source of NPN for ruminants (Nolan et al. 2010; Li et al. 2012)
and improve microbial N outflow (Guo et al. 2009).
1.7 Hypothesis of the research
The majority of the world ruminant animals graze forage that contains insufficient
protein for optimal animal production. Elimination of rumen protozoa from ruminants
can increase the efficiency of nutrient utilisation, especially of protein. Importantly, this
increase in livestock productivity could occur alongside a reduction in CH4 emissions.
Supplementation of fatty acids or dietary NO3 shows the persistent efficacy of CH4
mitigation, but these CH4 mitigations may suppress animal intake and productivity, and
NO3 supplement cannot currently be used as a practical means because of potential risk
of nitrite toxicity to the host. Therefore, this research was conducted to test the
hypothesis that elimination of rumen protozoa from ruminants and combination of
defaunation with fatty acids or NO3 supplementation could increase the productivity of
ruminants, while reducing the amount of CH4 emitted.
To address the hypothesis, the experimental program conducted is presented in the
following order in this thesis: While studies were made in both sheep and cattle, this
was not with intent to compare effects of defaunation cross species but simply to use the
Chapter 1: Literature review
29
most relevant species for each issue considered to be critical to advancing defaunation
towards a practical means of improving ruminant productivity.
- Chapter 2 as a baseline study will confirm the efficacy of defaunation to reduce
CH4 emissions and increase productivity of sheep in a controlled feeding
environment.
- Chapter 3 will examine the application of defaunation in the grazing
environment and determine CH4 emissions while grazing.
- Chapter 4 will examine the scope of combining defaunation and NO3
supplementation to enhance productivity and suppress CH4 emissions of sheep
offered low quality roughage.
- Chapters 5 and 6 will examine the effect of defaunation and NO3 (in vitro) and
confirm effects of defaunation and fatty acids (in vivo) on reducing CH4
emissions in beef cattle.
- And Chapter 7 in recognition of the difficulty experienced in defaunating cattle,
seeks to understand the role of retained protozoa in other stomachs by
quantifying the scale of sequestration of protozoa in the rumen, reticulum and
omasum.
Chapter 2: Refaunation of sheep after being protozoa-free
31
Chapter 2
Methane emissions, ruminal characteristics and
nitrogen utilisation changes after refaunation of
protozoa-free sheep
S. H. Nguyena, c,
, G. Bremnera, M. Cameron
b, R. S. Hegarty
a
a School of Environmental and Rural Science, University of New England, Armidale,
NSW 2351, Australia
b NSW Department of Primary Industries, Armidale, NSW 2351
c National Institute of Animal Sciences, Hanoi, Vietnam
Small Ruminant Research, 2016, 144. 48-55
Chapter 2: Refaunation of sheep after being protozoa-free
32
Abstract
Effects of rumen protozoa on ruminal fermentation, methane (CH4) emissions and
nitrogen (N) retention were studied in twelve crossbred ewes given an oaten chaff diet.
Over 10 days sheep were progressively adapted to a diet containing 7% coconut oil
distillate to suppress rumen protozoa and then were defaunated using sodium 1-(2-
sulfonatooxyethoxy) dodecane (Empicol). Twelve weeks after defaunation treatment,
five sheep were inoculated with rumen fluid collected from cannulated sheep to
refaunate them and the effect of re-establishment of rumen protozoa 0, 7, 14 and 21
days following refaunation on ruminal fermentation and CH4 emissions was then
examined in Experiment 1. As a following study (Experiment 2), feed intake was
restricted to 1.5 × ME maintenance from day 28 to day 43 when dry matter digestibility
(DMD), N retention, fermentation and daily methane production (DMP) were compared
between defaunated and refaunated sheep in a randomised block design. Sheep were
scanned by a computed tomography scanner on day 0 and day 28 to estimate reticulo-
rumen (RR) weight and carcass composition. It was concluded that refaunated sheep did
not have a higher daily DMP than did the defaunated cohort within 21 days after
refaunation as measured by GreenFeed Emission Monitoring units. Total volatile fatty
acid (VFA) concentration and the proportion of propionate in the rumen VFA gradually
increased over 21 days following refaunation (Experiment 1), while a change towards
higher butyrate and lower acetate proportions was observed after 28 days (Experiment
2; P < 0.05). In experiment 2 feed intake was fixed for comparative studies. There was a
tendency towards a heavier RR weight (P = 0.08) and a higher ratio of RR to liveweight
in defaunated sheep 28 days after refaunation (P < 0.001), but carcass composition was
Chapter 2: Refaunation of sheep after being protozoa-free
33
not affected by refaunation status. Experiment 2 showed defaunated sheep had a 7%
lower DMP and methane yield (g CH4/kg DMI) than did refaunated sheep with an
established rumen fauna (P < 0.05). Apparent whole-tract N digestibility, DMD and
microbial crude protein supply were not different between defaunated and refaunated
sheep, while energy losses in CH4 (MJ/d) and CH4 as a proportion of gross energy
intake were both approximately 8% lower in defaunated sheep.
Keywords: Ciliates, methanogens, carcass composition.
2.1 Introduction
Rumen protozoa are important, but not essential in the rumen ecosystem and for the
well-being of host animals (Williams and Coleman 1992). Elimination of rumen
protozoa (defaunation) has led to reported increases in growth rate and average daily
gain (ADG) of ruminants (Bird et al. 1979; Eugène et al. 2004a; Newbold et al. 2015)
especially when the feed is deficient in protein relative to energy content with respect to
the animal’s requirements. In addition, rumen protozoa are significant hydrogen (H2)
producers and synthesise mainly acetate and butyrate rather than propionate, so
defaunation may be expected to induce a greater proportion of propionate in the ruminal
VFA, but this phenomenon is not always observed (Williams and Coleman 1992;
Hegarty et al. 2008). The reduced daily methane production (DMP) associated with
defaunation reported by several authors (Hegarty 1999; Eugène et al. 2004a; Newbold
et al. 2015) may also reflect a reduced ruminal H2 availability due to (1) reduced acetate
and H2 production, (2) increased H2 use in propionic acid synthesis, (3) reduced
endosymbiotic methanogens associated with rumen protozoa (Finlay et al. 1994; Tokura
Chapter 2: Refaunation of sheep after being protozoa-free
34
et al. 1997) and (4) decreased total ruminal dry matter fermentation. Despite these
potential actions, defaunation did not change enteric CH4 production 10 to 25 weeks
post-treatment (Bird et al. 2008) and did not affect CH4 production by lambs without
protozoa from birth or from weaning (Hegarty et al. 2008). The absence of rumen
protozoa, therefore, does not always reduce DMP (Morgavi et al. 2012; Kumar et al.
2013). So, the role of rumen protozoa in moderating the overall H2 economy and
fermentation in the rumen is not consistent or clearly understood. This study aimed to
examine the time-course of protozoa establishment, fermentation, intake and CH4
production changes that occur following refaunation of the rumen (Experiment 1) and
then to assess fermentation, CH4 production, reticulo-rumen characteristics and nitrogen
retention and digestibility in defaunated and refaunated sheep with a stable rumen
ecology (Experiment 2). This was done to better understand the contribution (direct or
indirect) of ciliate protozoa to rumen metabolism and methanogenesis.
2.2 Materials and methods
2.2.1 Preparation of defaunated animals
All protocols for the care and treatment of the sheep were approved by the University of
New England Animal Ethics Committee (AEC 14-083). Twelve crossbred ewes (Border
Leicester rams × Merino ewes) about 24 months of age with an average liveweight (±
s.e) of 55 ± 4 kg were selected and acclimated to a diet of blended lucerne and cereal
(LC) chaff for three days while being held in individual pens, so they had no direct
physical contact with each other. Sheep were then supplemented with dietary coconut
oil distillate (COD; PT Nuansa Kimia Sejati, Tangerang, Indonesia.), from an initial
Chapter 2: Refaunation of sheep after being protozoa-free
35
inclusion of 3% progressing to 7% COD inclusion in the diet (as fed) over 10 days to
supress rumen protozoa. The diet of COD was prepared by sprinkling the liquid COD
onto LC while the chaff was tossed in a rotary feed mixer. After 10 days of COD
inclusion, sheep were fasted and orally dosed with sodium 1-(2-sulfonatooxyethoxy)
dodecane (Empicol ESB/70AV, Albright and Wilson Australia Ltd, Melbourne)
administered at 10 g/day in a 10% v/v solution for three consecutive days to remove
protozoa, with feed being withheld during this period. This defaunation protocol was
adapted from Bird and Light (2013). Chaff was offered to defaunated sheep 5 hours
after the last dose of Empicol. One defaunated sheep was removed from the experiment
due to inappetence, but other sheep recovered their pre-treatment voluntary intake
within 11 days post-dosing. Small entodiniomorph protozoa were found in some rumen
fluid samples 11 days after the treatment completed, so defaunated sheep were again
supplemented with 7% COD for 6 days, fasted and dosed for two days with Empicol as
before, with feed being withheld during dosing. After the second defaunation treatment,
sheep were offered LC and maintained in individual pens for 2 weeks then rumen-
samples collected and examined to ensure that rumen protozoa could not be visually
detected in any rumen fluid before sheep were moved into 2×2 ha paddocks which
excluded their contact with other ruminants (Table 2.1)
After ten weeks of recovery following defaunation treatment, sheep were randomly
allocated into 2 groups by stratified randomisation by ranking on liveweight then
randomly allocated to treatment within 6 weight classes and housed in two separate
group pens in an animal house. Sheep were offered ad libitum access to oaten chaff
(OC; 9.5 MJ/kg DM and 7.3% CP) delivered by automatic feeders and a restricted pellet
Chapter 2: Refaunation of sheep after being protozoa-free
36
supplement (12 MJ/kg DM and 16.8% CP) was delivered by GreenFeed Emission
Monitoring (GEM) units (one GEM per pen; Table 2.2). Radio-frequency identification
(RFID) sensors were fitted in both the automatic chaff dispensers and GEM to identify
and record individual animal intakes. After 2 week acclimation to the automatic feeders
and GEM, Experiment 1 commenced (day 0, Table 2.1).
Table 2.1 Experimental schedule for the defaunation, refaunation and data
measurements.
Day Activity
Defaunation period
-113 Coconut oil distillate (7% COD) feeding period
-103 First dosing protocol and recovery period
-90 COD feeding period between 2 dosing protocol
-84 Second dosing protocol and recovery period
Recovery period
-71 Defaunated sheep were placed in an isolated paddock for grazing
-15 Defaunated sheep were allocated to defaunated and refaunated groups
and fed oaten chaff in 2 separate group pens. Methane emissions were
measured by GEM and intake was measured by auto-feeders.
-1 Defaunated sheep were scanned to determine RR volumes by CT
scan, liveweight was measured before scanning
Refaunation and
measurement period
0 Rumen fluid collection for initial measurements. Refaunated 5 sheep
with fresh rumen fluid from cannulated sheep for 2 days (50 mL per
sheep.day)
7 Rumen fluid, liveweight and methane emissions from green-feeds
were collected every week commencing on d 0, 7, 14 and 21.
28 Sheep were scanned for the last measurement of RR volumes by CT
scan
29-38 Total collection period
39-43 Sheep were placed in respiration chambers to measure methane
emissions.
Chapter 2: Refaunation of sheep after being protozoa-free
37
Table 2.2 Chemical composition of the pellets supplied through the Greenfeed Emision
Monitoring (GEM) unit and of oaten chaff (g/100g dry matter)
Component GEM
pellets
Oaten
chaff
Dry matter (in feed as-fed) 90.1 89.5
Dry matter digestibility 75 65
Digestible organic matter 75 62
Inorganic ash 9 7
Organic matter 91 93
Neutral detergent fibre 31 55
Acid detergent fibre 10 29
Crude protein 16.8 7.3
Crude fat 3.9 -
Metabolisable energy (MJ/kg DM) 12.1 9.5
2.2.2 Experiment 1
Starting on day 0, one group of defaunated sheep (n=5) was inoculated (50 mL per
sheep per day on 2 consecutive days) with fresh mixed rumen fluid collected from 5
rumen cannulated sheep fed roughage. Samples of rumen fluid (20 mL) were then
collected from each sheep weekly by oesophageal intubation with a fresh collection tube
used for each animal for protozoal enumeration, volatile fatty acid (VFA) and ammonia
(NH3) analyses. Rumen pH was measured immediately using a portable pH meter
(Orion 230 Aplus, Thermo scientific, Beverly, MA, USA). A 15 mL subsample was
placed in wide-neck McCartney bottle acidified with 0.25 mL of 18 M sulphuric acid
Chapter 2: Refaunation of sheep after being protozoa-free
38
and stored at -200C for VFA and NH3 analyses. A 4 mL subsample was placed in wide-
neck McCartney bottle containing 16 mL of formaldehyde-saline (4% formalin v/v) for
protozoa enumeration. Protozoa were counted using a Fuchs–Rosenthal optical counting
chamber (0.0625 mm2 and 0.2 mm of depth) using a staining technique adapted from
the procedure of Dehority (1984). The protozoa were differentiated into large (>100
µm) and small (<100 µm) holotrich and entodiniomorph groupings. The VFA
concentrations were determined by gas chromatography (Nolan et al. 2010) using a
Varian CP 3800 Gas Chromatograph (Varian Inc. Palo Alto, California USA) and NH3
concentration was analysed by a modified Berthelot reaction using a continuous flow
analyser (San++
, Skalar, Breda, The Netherlands).
2.2.3 Methane measurement by GreenFeed Emission Monitoring units
Immediately following refaunation, DMP of all sheep was monitored by the GEM unit
present in each pen. For each week, DMP of each sheep was averaged for the 7 days
prior to day 0, 7, 14 and 21 and together with the sum of dry matter intake (DMI) in OC
and pellets in each period was used to calculate CH4 yield (MY; g CH4/kg DMI). For
CH4 to be measured in the GEM, sheep voluntarily placed their heads in a shroud and
were detected by the RFID sensors, triggering the GEM to progressively release pellets
(Hammond et al. 2016). Eructated CH4 was measured while sheep consumed the pellet
supplement. Pellets were dispensed to individual sheep at a minimum of every 4
h/supplementation event (total maximum of 6 supplementation events per day). At each
supplementation event, up to 5 drops of pellets were made, with drops being made at
40s intervals and providing 7.74 ± 0.54 g pellets/drop. This supplementation regime
Chapter 2: Refaunation of sheep after being protozoa-free
39
routinely ensured sheep stayed at the GEM for CH4 and carbon dioxide (CO2) flux
measures for at least 2 min while being supplemented. All visits to the autofeeders and
GEM units were continuously recorded from 7 days prior to day 0 and through to day
21. Sheep were also measured for liveweight weekly in the morning without fasting.
2.2.4 Experiment 2
Samples of rumen fluid (20 mL) were collected from each sheep on days 30 and 38 for
protozoal enumeration, VFA and NH3 analyses with sampling procedures as described
in Experiment 1. From day 28, sheep were restricted fed at 1.5 × ME maintenance for 4
days before being moved into individual metabolism cages to conduct a 5-day total
collection of excreta and then DMP was measured in open circuit respiration chambers.
The maintenance requirement (MEm; MJ ME/d) was calculated from the Australian
feeding standards (CSIRO 2007). The reason for restricting feed intake was to allow
comparison of DMP and N retention of defaunated and refaunated sheep without
confounding by variable daily DMI. The average liveweight of sheep on day 28 was
used to calculate ME requirement for all sheep for the restricted feeding period (1.5 Mm)
which was an average of 68% of their previous ad libitum intake. Feed was divided in
two equal portions and offered at 1000 hours and 1500 hours daily during the total
collection period. Sheep had free access to fresh water renewed every morning in a
water trough.
Chapter 2: Refaunation of sheep after being protozoa-free
40
2.2.5 Estimation of reticulo-rumen weight, gas proportion and
carcass composition
On day 0 and 28 after protozoa inoculation, a whole body scan from 3rd
- 4th
thoracic
vertebrae and 1st - 2
nd caudal vertebrae with 5 mm thickness, 10 mm spacing and 480
mm field of view was performed using a Picker UltraZ 2000 Computed Tomography
(CT) scanner, Philips (Philips Medical Imaging Australia, Sydney, NSW) as described
by Kvame and Vangen (2007). After scanning, each CT image was edited using the
software program OsiriX (Rosset et al. 2004) to estimate reticulo-rumen (RR) volume
and then remove non-carcass tissues from each image, leaving carcass fat, lean and
bone for estimation of carcass weight (CW) and composition. These images were
further divided into tissue areas of fat (-194 to -23), lean (-22 to 146) and bone (147 to
1024) in Houndsfield Units (HU) using the ImageJ software program, which was
developed on methods similar to those described by Thompson and Kinghorn (1992)
and these were corrected for tissue density to provide estimates of tissue weight based
on the relationship between HU and density (Fullerton 1980). Rumen gas volume was
calculated by the difference between RR volume estimated by OsiriX with air and
estimated by ImageJ without air, and gas proportion was calculated as the gas volume
expressed as proportion of the total RR volume.
2.2.6 Nitrogen digestibility, energy utilisation, and microbial protein
outflow
A 5-day collection of faecal and urinary output was conducted from day 33 to day 38.
All excreta output over the 5 days was collected and weighed, with feed DMI and faecal
Chapter 2: Refaunation of sheep after being protozoa-free
41
DM output used to determine DMD. Dry matter content of feed and faeces were
determined by drying samples at 60°C in a fan-forced oven to a constant weight. The
concentration of allantoin in the urine was determined colorimetrically (IAEA 1997)
using a UV-1201 spectrophotometer (Shimadzu, Japan) reading at 522 nm. The yield of
total microbial crude protein (MCP) outflow from the rumen was calculated from
allantoin output by using the equations of Chen et al. (1992).
Total nitrogen (N) in feed, faeces and urine were determined using an automated
Organic Nitrogen Determinator (FP-2000, Leco Corporation, St Joseph, MI). Gross
energy (GE) content of feed, faeces and urine were determined using a bomb
calorimeter (Calorimeter C7000 with cooling system C7002, IKA Werke, Germany).
The energy loss through CH4 was calculated assuming a CH4 energy density of 55.6
MJ/kg (Bossel and Eliasson 2003). The ME was determined from the GE consumed less
the measured energy loss through faeces, urine and CH4.
2.2.7 Methane measurement by respiration chambers
Daily methane production was measured by open-circuit respiration chambers from day
39 to day 43 using 2×22 h consecutive periods per sheep (Bird et al. 2008). Defaunated
sheep were first measured from day 39 to day 41 and refaunated sheep were measured
from day 41 to day 43 to avoid the risk of protozoal cross-contamination. Sheep were
placed in individual respiration chambers by 1100 hours, with their feed and water
already available inside the chambers. The chambers were opened to collect feed
refusals, clean faecal trays, and supply fresh feed and water at 0900 hours the following
day and then were resealed at 1100 hours. Concentration of CH4 of air leaving the
Chapter 2: Refaunation of sheep after being protozoa-free
42
chamber was measured by a photoacoustic gas analyser (Innova Model 1312, AirTech
Instruments, Ballerup, Denmark). Recovery of CH4 through the chambers was
determined by injection of a known volume of CH4 and measurement of CH4
concentration every 2 min for 20 min, with recovery of the dose being calculated by
integrating the area under the concentration curve over time.
2.2.8 Statistical analyses
Data was statistically analysed using SAS 9.0 (SAS Institute, Cary, NC). Fermentation
parameters, DMI, DMP and liveweight collected in Experiment 1 were subject to
repeated-measures analysis of variance in PROC MIXED with protozoal treatment, day
and protozoa × day interaction as fixed factors. Fermentation parameters, DMP, whole-
tract DMD and N retention collected in Experiment 2 were subject to analysis of
variance in PROC GLM. For comparison of RR parameters and carcass composition of
defaunated and refaunated sheep on day 28, the model used initial RR parameters and
carcass composition on day 0 as a covariate. Protozoa count was log-transformed before
statistical analysis. Homogeneity of variance and normal distribution were tested using
PROC UNIVARIATE before statistical analysis. Means were analysed using the least
squares means (LSMEANS) procedure. A probability of error of less than 5% was
considered to be statistically significant.
Chapter 2: Refaunation of sheep after being protozoa-free
43
2.3 Results
2.3.1 Experiment 1
2.3.1.1 Rumen protozoal establishment in refaunated sheep
The concentration of total protozoa in the rumen liquor before defaunation commenced
was 17.01 ± 5.88 ×105 cells/mL of which small entodiniomorphs accounted for 97% of
the total protozoa. Rumen protozoa were reduced by 98% (data not shown) after 10 day
supplementation with 7% COD, with holotrichs not visually detected after this time.
Sheep became protozoa-free after two programs of treatment with Empicol and stayed
free throughout the experiment unless protozoa were introduced for refaunation. The
protozoal population in the refaunation inoculum was 11.20 × 105
cells/mL with small
entodiniomorphs being 80% of the total protozoa. After inoculation, the protozoal
population of refaunated sheep reached 12.94×105 cells/mL by day 21 which was
similar to the protozoal population in the inoculum (P > 0.05) before settling to
9.06×105 cells/mL on day 30 (P < 0.05; Figure 2.1). Small entodiniomorphs were
predominant, but decreased from 93% to 80% of the total population from day 7 to 38,
respectively while the population of large (> 100 μm) protozoa increased over this time
from 4.9% to 18% (Figure 2.1).
Chapter 2: Refaunation of sheep after being protozoa-free
44
Figure 2.1 Protozoal population of the inoculum and of refaunated sheep on day 7, 14,
21, 30 and 38 after protozoa inoculation. Columns indicate total protozoal population
(cells/ml). (□) indicates small entodiniomorphs (< 100 μm). (■) indicates large protozoa
(> 100 μm). Error bars indicate pooled s.e of total protozoa. Restricted feed intake
occurred after day 21.
2.3.1.2 Ruminal fermentation
Refaunated sheep had higher rumen pH and NH3 concentration than did defaunated
sheep on day 7 and up to day 21 after refaunation (P < 0.05; Table 2.3). The
introduction of rumen protozoa increased total VFA concentration and the proportion of
propionate while the acetate to propionate ratio was lower in refaunated sheep (P <
0.05). The proportion of acetate was not affected by the presence or absence of rumen
protozoa (P > 0.05), but did change with day, being lower 21 days after refaunation
while the proportion of butyrate was affected by neither the presence of rumen protozoa
nor by day after refaunation (P > 0.05).
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
Inoculum Day 7 Day 14 Day 21 Day 30 Day 38
Pro
tozo
al p
op
ula
tio
n (
×1
0⁵/
mL
)
Chapter 2: Refaunation of sheep after being protozoa-free
45
Table 2.3 Weekly rumen fermentation characteristics, intake and methane emission of defaunated (-P) sheep and of refaunated (+P) sheep before
(Day 0) and up to 21 days after protozoa inoculation.
Parameter
-P (n = 6)
+P (n = 5)
Pooled
s.e
P-value
Day 0 Day 7 Day 14 Day 21 Day 0 Day 7 Day 14 Day 21 Protozoa
effect Day
effect Protozoa × day
effect
Liveweight (kg) 54.65 56.08 56.92 57.83 55.18 57.36 57.40 57.86 2.13 0.85 <0.001 0.41
Rumen pH 6.45 6.50 6.33 6.43 6.42 6.62 6.60 6.58 0.05 0.004 0.09 0.15
NH3-N (mg/L) 55.9 52.38 55.62 50.72 59.54 65.94 62.83 65.24 4.21 0.01 0.98 0.49
Total VFA (mM/L) 90.65 88.29 87.83 88.10 88.49 100.96 103.71 101.88 5.02 0.01 0.49 0.12
Acetate (molar %) 69.80 68.93 67.32 68.26 69.79 68.22 64.35 63.12 1.29 0.10 0.002 0.11
Propionate (molar %) 16.68 16.54 16.92 16.16 16.23 21.80 23.95 23.53 0.82 <0.001 <0.001 0.001
Butyrate (molar %) 9.33 10.31 10.52 10.11 10.53 9.48 10.36 10.98 0.40 0.37 0.25 0.06
Acetate: propionate ratio 4.20 4.20 4.02 4.28 4.36 3.17 2.73 2.73 0.21 0.003 0.004 0.009
Chaff DMI (g/day) 1365 1497 1423 1379 1428 1517 1416 1396 7.25 0.005 0.001 0.009
Pellet DMI (g/day) 162.6 161.2 165.4 163.6 150.1 165.6 181.6 172.9 6.00 0.59 <0.001 0.001
Total DMI (g/day) 1528 1658 1588 1542 1578 1683 1579 1569 8.90 0.01 0.001 0.14
Pellet drops/day 26.71 26.48 27.17 26.88 24.11 26.60 29.17 27.77 0.95 0.94 <0.001 <0.001
DMP (g CH4/day)†
27.83 31.83 33.16 35.17 27.00 31.40 33.60 37.40 1.76 0.88 <0.001 0.06
MY (g CH4/kg DMI)†
18.22 19.19 20.88 22.79 17.12 18.70 21.02 23.84 0.95 0.90 <0.001 0.02
†Daily methane production (DMP) and methane yield (MY) measured by the GEM unit; Dry matter intake (DMI).
Chapter 2: Refaunation of sheep after being protozoa-free
46
2.3.1.3 Dry matter intake and methane emissions
The presence of rumen protozoa increased DMI (P = 0.01; Table 2.3) with ad libitum
intake of oaten chaff being higher in refaunated sheep (P < 0.01), while the pellet
intake, which was mechanically regulated, was unchanged by refaunation, but was
affected by day. Defaunated and refaunated sheep received a similar number of pellet
drops per day (P > 0.05) which were close to the maximum allocation of 30 drops per
sheep per day, Defaunated and refaunated sheep did not differ in DMP or MY (P >
0.05), but these increased from day 0 to day 21 for both treatments with 21% and 28%
increased DMP and 20% and 28% increased MY in defaunated and refaunated sheep,
respectively (P < 0.001; Table 2.3). Liveweight did not differ between protozoal
treatments (P > 0.05), but increased by 5.5% and 4.6% from day 0 to day 21 in
defaunated and refaunated sheep, respectively (P < 0.001; Table 2.3).
2.3.2 Experiment 2
2.3.2.1 Estimation of reticulo-rumen weight and carcass composition
The presence of rumen protozoa 28 days after refaunation tended to decrease RR weight
(including tissue and content; P = 0.08) and significantly decreased the ratio of RR to
liveweight (P < 0.001), but did not change RR volume, gas volume or proportion of gas
space in the RR as estimated by CT scan (P > 0.05; Table 2.4). There was a strong
positive correlation between RR weight and RR volume (RR weight = 0.94 RR volume
– 0.058; r2 = 0.96; P < 0.001; Figure 2.2). Carcass weight and weight of fat, lean and
Chapter 2: Refaunation of sheep after being protozoa-free
47
bone 28 days after refaunation were not affected by the presence or absence of rumen
protozoa (P > 0.05).
Table 2.4 Reticulo-rumen (RR) volume, weight, gas volume and gas proportion of
defaunated (- P) sheep and of refaunated (+ P) sheep. Day 0 data was used as a
covariate.
Parameter
-P (n = 6) +P (n = 5)
Pooled s.e P-value
Day 0† Day 28 Day 0
† Day 28
RR volume (cm3)
10591 10661 11170
9923
365 0.19
RR weight (kg) 9.75
10.12
10.55
9.16
3.32 0.08
Gas volume (cm3) 1373 1241 1199 1498 236 0.47
Gas proportion (%) 12.9 11.81 10.78 14.93 2.1 0.33
RR/LW ratio (%) 17.84
17.95
18.99
15.92
0.65 < 0.01
Estimated CW (kg)‡
27.11 30.84 26.49 33.88 1.46 0.18
Carcass fat (kg) 6.38 7.96 6.03 8.95 0.61 0.28
Carcass lean (kg) 17.16 19.20 16.96 21.05 0.80 0.14
Carcass bone (kg) 3.57 3.68 3.50 3.89 0.09 0.14
Fat as a % of CW‡
23.45 25.51 22.68 26.43 0.83 0.46
Lean as a % of CW‡ 63.29 62.45 64.11 62.03 0.60 0.64
Bone as a % of CW‡ 13.26 12.2 13.21 11.58 0.33 0.37
†Day 0 data was used as a covariate and statistical comparison were between day 28. ‡Carcass weight (CW).
Chapter 2: Refaunation of sheep after being protozoa-free
49
Table 2.5 Rumen fermentation characteristics, feed intake, methane emission and
nutrient utilisation of defaunated (- P) sheep and of refaunated (+ P) sheep offered a
fixed intake (Experiment 2).
Parameter Protozoal treatment
Difference (-P) - (+P)
with pooled s.e P-value
-P (n=6) +P (n=5)
pH 6.45 6.76 -0.31± 0.14 0.17
NH3-N (mg/L) 44.82 60.37 -15.55 ± 3.61 0.01
Total VFA (mM/L) 56.14 54.19 +1.95 ± 12.13 0.92
Acetate (molar %) 74.70 69.35 +5.35 ± 0.95 0.04
Propionate (molar %) 14.98 16.78 -1.8 ± 0.26 <0.01
Butyrate (molar %) 6.86 9.03 -2.17 ± 0.36 0.002
Acetate/propionate ratio 4.99 4.14 +0.85 ± 0.14 0.001
DMP (g CH4/day)†
19.57 21.03 -1.46 ± 0.47 0.04
MY (g CH4/kg DMI)†
18.44 19.85 -1.41 ± 0.43 0.03
ADG (g/day)‡ 59.92 57.62 +2.3 ± 14.00 0.91
DMD (%)§
59.16 61.21 -2.05 ± 0.95 0.16
Faecal N (g/day) 8.33 8.40 -0.07 ± 0.38 0.90
Urinary N (g/day) 7.45 6.85 +0.60 ± 1.31 0.75
N retention (g/day) 8.91 9.44 -0.53 ± 1.11 0.74
Apparent N digestibility (%) 66.24 65.98 +0.30 ± 1.55 0.91
MCP supply (g/day)¶ 6.28 5.37 +0.91 ± 0.91 0.49
Energy loss in faeces (MJ/day) 8.178 7.769 +0.41 ± 0.17 0.13
Energy loss in urine (MJ/day) 0.438 0.398 +0.04 ± 0.06 0.66
Energy loss in CH4 (MJ/day)
1.04 1.13 -0.09 ± 0.03 0.04
Energy loss in CH4/GE intake (%) 5.61 6.07 -0.46 ± 0.15 0.04
ME (MJ/day) 8.94 9.24 -0.30 ± 0.19 0.22
† Daily methane production (DMP) and methane yield (MY) measured by respiration chambers during fix intake
period; ‡Average daily gain (ADG); §Dry matter digestibility (DMD); ¶Microbial crude protein (MCP)
Chapter 2: Refaunation of sheep after being protozoa-free
50
After day 28 when the feed offer was offered at a restricted and uniform daily rate for
digestibility and respiration chamber studies, all feed offered was consumed by both
defaunated and refaunated sheep with average GE and N intakes of 18.6 MJ/day and
24.7 g N/day (data not shown), respectively. Refaunation did not significantly change
the whole-tract DMD, N digestibility, N retention or ME available (P > 0.05; Table
2.5). Outflow of MCP was approximately 15% greater in defaunated than refaunated
sheep, but there was not a statistical difference (P > 0.05). By this time after
refaunation, refaunated sheep had a stabilised rumen fermentation with no difference in
total VFA concentration, but a greater proportion of propionate and also increased DMP
and MY compared to defaunated sheep. Refaunation significantly increased energy loss
in CH4 (MJ/day, P = 0.04) as determined by respiration chambers by approximately 8%
and increased energy loss as a percentage of GE intake (7.6%; P = 0.04) due to the 7%
increased DMP with refaunation.
2.4 Discussion
2.4.1 Protozoal population in refaunated sheep after inoculation
The protozoal population in sheep after refaunation from a previously defaunated state
was well established by day 7 and reached 12.9×105 cells/mL by day 21, comparable
with that found by Morgavi et al. (2008) who demonstrated that total protozoal
populations reached their peak at 12×105
cells/mL by 25 to 30 days after inoculation,
then stabilised at a slightly reduced population (7.6×105
cells/mL). Sénaud et al. (1995)
also re-inoculated the defaunated rumen with Isotricha sp. alone or mixed ciliates and
reported that the maximum concentration of rumen protozoa was reached 9 to 17 days
Chapter 2: Refaunation of sheep after being protozoa-free
51
after inoculation. The maximum population then decreased for 2-3 days before
stabilising. Further, Zeitz et al. (2012) who examined individual growth of Entodinium
caudatum, Epidinium ecaudatum or Eudiplodinium maggii found that a stabilised
population size was reached between 2 and 4 weeks after protozoa inoculation.
2.4.2 Ruminal fermentation and microbial protein outflow
Refaunation increased rumen pH within 21 days of inoculation, which is consistent with
previous assessments (Williams and Coleman 1992; Machmüller et al. 2003; Eugène et
al. 2004a), but did not differ from the defaunated group in the restricted feeding period
(Newbold et al. 2015). Rumen protozoa are able to metabolise lactic acid (Williams and
Coleman 1992), so reduce the risk of acidosis associated with a sudden fall in ruminal
pH (Jouany and Ushida 1999). Franzolin and Dehority (2010) reported that rumen pH
was higher in faunated steers compared to defaunated ones with the mean pH values of
5.98 and 5.50, respectively. These authors also observed that rumen pH was highest
before feeding, but lower 4 h after feeding in both defaunated and faunated steers.
The greater proportion of propionate in refaunated sheep by 21 days after inoculation
and the higher proportion of propionate, and lower proportion of acetate subsequently
observed in refaunated sheep under the restricted feeding condition were inconsistent
with previous findings of ciliate protozoa being significant producers of H2 and
synthesising acetic and butyric acids rather than propionic acid (Williams and Coleman
1992). Defaunation is also generally associated with fermentation shifting to a greater
proportion of propionic acid, therefore reducing the amount of CH4 produced (Eugène
et al. 2004a). However, this phenomenon is not always observed (Williams and
Chapter 2: Refaunation of sheep after being protozoa-free
52
Coleman 1992). A decreased proportion of propionate (Machmüller et al. 2003; Hegarty
et al. 2008) and a higher proportion of acetate in the VFA of defaunated animals has
being reported (Bird 1982) when animals were fed roughage based diets. An increased
acetate proportion with defaunation may be explained by stoichiometric laws by
Sauvant et al. (2011), although low digestibility diets typically cause a higher acetate
production accompanied by lower ATP yield. A recent meta-analysis by Newbold et al.
(2015) also found defaunation substantially decreased butyrate and increased acetate,
but did not affect propionate proportion.
The finding that ruminants with protozoa (refaunated) supported a higher ruminal NH3
concentration than did animals with rumen protozoa removed (defaunated) in this study
indicated that defaunation may allow a proliferation of rumen bacteria to increase
uptake of NH3 by bacteria for protein synthesis as well as less protein being degraded in
the absence of protozoa (Jouany and Ushida 1999). After refaunation, rumen NH3
concentration increased (Table 2.2) and was 26% higher in refaunated than defaunated
sheep after 28 days (Table 2.4), confirming previous assessments (Eugène et al. 2004a;
Morgavi et al. 2012; Newbold et al. 2015). Less ruminal catabolism of feed-protein and
bacteria occurs in the absence of protozoa, usually leading to an increase in the supply
of protein to the duodenum (Bird and Leng 1978; Jouany 1996) and the increased MCP
outflow is associated with 9-35% increased ADG in defaunated animals given forage
diets (Bird 1989). The present study showed an approximate 15% increase in MCP
outflow in defaunated relative to refaunated sheep with a 4% increased ADG (Table
2.4).
Chapter 2: Refaunation of sheep after being protozoa-free
53
2.4.3 Methane emissions
Sequential DMP measures made by the GEM for up to 21 days after protozoa
inoculation were not different from those of defaunated sheep. This confirms an earlier
study by Morgavi et al. (2008) in which refaunated wethers had a similar DMP as
defaunated wethers 4 weeks after protozoa inoculation, although protozoal numbers
were comparable with conventional animals. Monitoring DMP over 21 days in
defaunated and refaunated sheep found that DMP was gradually increased over time in
both treatments, indicating that rumen microbes in defaunated sheep had probably not
been stabilised after 12 weeks of defaunation treatment and/or the absence of rumen
protozoa induced the increased microbial H2 producers such as cellulolytic ruminococci
(Mosoni et al. 2011). The increased CH4 emissions of 7% were observed later in the
refaunation treatment at day 43 (Table 2.4). This may be partly explained by the
increased proportion of large protozoa in the total protozoal count (Figure 2.1),
increasing the butyrate production and H2 availability for CH4 formation (Williams and
Coleman 1992). Large protozoa also contribute considerable amounts of formate
(Tokura et al. 1997), which is a substrate for methanogenesis (Leng 2014).
A significant reduction of DMP has been reported in defaunation of animals fed high
quality forage or a grain-based diet in association with the increased propionate
production (Whitelaw et al. 1984; Kreuzer et al. 1986; Eugène et al. 2004a; Mosoni et
al. 2011), but differences in DMP were much smaller or even not significant between
defaunated and faunated animals on a forage-based diet, on which no increase in
Chapter 2: Refaunation of sheep after being protozoa-free
54
propionate proportion was observed (Hegarty et al. 1994; Ranilla et al. 2007; Bird et al.
2008; Hegarty et al. 2008).
The lower DMP by defaunation of animals is probably related to higher concentration
of dissolved H2 due to the reduced capacity to utilise H2 by rumen microbes (Janssen
2010; Morgavi et al. 2012) after removing the endo-symbiotic and ecto-symbiotic
methanogens associated with rumen protozoa (Finlay et al. 1994; Tokura et al. 1997).
Ruminal acetogens were found to grow on CO2 and H2, and produce acetate, but
acetogenesis is generally thought to not occur in the rumen because of the higher H2
threshold and lower H2 affinity of acetogens compared to methanogenesis (Joblin
1999). In normal fermentation, methanogens reduce H2 to a low level in which
reductive acetogenesis is below detectable levels (Ungerfeld 2015), but if acetate is
produced when methanogenesis is inhibited, H2 may have accumulated, stimulating
reductive acetogenesis (Ungerfeld 2013). Reductive acetogens established in the rumen
lacking methanogens can replace methanogens as a sink for H2 in the rumen (Fonty et
al. 2007). Although H2 concentration was not measured in this study to test this
hypothesis, the reduced CH4 emissions from defaunated sheep in this study associated
with a rise in acetate proportion is consistent with the accumulation of H2 resulting from
reductive acetogenesis.
2.4.4 Reticulo-rumen weight and carcass composition
An established protozoal population (at 28 d) led to an approximately 10% smaller RR
weight and RR weight as a proportion of the liveweight compared to in defaunated
sheep. This was consistent with Orpin and Letcher (1984) who reported a 30%
Chapter 2: Refaunation of sheep after being protozoa-free
55
increased rumen volume associated with defaunation. Jouany et al. (1988) observed the
change in rumen volume following defaunation resulted from changes in ruminal
digestion. The increased weight of ruminal contents after defaunation was probably due
to longer particle retention of ruminal digesta associated with the rumen fill effect of
lower organic matter (OM) digestibility (Eugène et al. 2004a). Defaunation of
ruminants can increase ADG by 11% because the absence of rumen protozoa allows a
compensatory increase in bacterial populations, leading to more microbial protein
outflow in defaunated compared to faunated animals (Eugène et al. 2004a). There is
little data on carcass composition consequences of defaunation with mixed responses
reported (Hegarty et al. 2000). However, defaunation as a mean to increase protein
supply for absorption did not significantly affect body weight or carcass composition of
sheep in this study.
2.4.5 Whole-tract dry matter digestibility, nitrogen and energy
utilisation
Numerous studies in the literature reported a negative effect of the absence of rumen
protozoa on ruminal fibre degradation (Eugène et al. 2004a; Eugène et al. 2010;
Newbold et al. 2015), probably due to the loss of protozoal fibrolytic activity.
Polysaccharidase activity was greater in the rumen of faunated animals (Santra and
Karim 2002; Eugène et al. 2004b), resulting in a positive effect of protozoa on ruminal
digestion. However, Williams and Withers (1993) observed inoculation with rumen
protozoa led to the lowest population of fibrolytic bacteria. Subsequently, major rumen
culturable cellulolytic bacterial species such as Fibrobacter succinogenes,
Chapter 2: Refaunation of sheep after being protozoa-free
56
Ruminococcus albus and Ruminococcus flavefaciens have been shown to exist in
increased populations in response to defaunation of the rumen (Mosoni et al. 2011;
Zeitz et al. 2012) due to less competition between bacteria and ciliate protozoa and less
predation by protozoa.
Defaunation often induces a shift of N excretion from urine to faeces, but this was not
observed in this study. Because the absence of rumen protozoa results in a 5-15%
reduction of ruminal digestion of carbohydrate of plant cell walls (Jouany et al. 1988)
which is compensated for a greater digestion in the large intestine (Ushida et al. 1991),
more microbial protein is formed in the large intestine, thus the increased faecal N loss
is observed (Jouany 1996). In addition, defaunation also causes lower urinary N
excretion due to the lower NH3 concentration in the rumen and less NH3 absorbed,
reducing hepatic urea-synthesis and urinary N loss.
The compensatory increase in cellulolytic bacteria (Mosoni et al. 2011) and the greater
digestion of carbohydrate of plant cell walls in the large intestine (Ushida et al. 1991)
that is often associated with the absence of rumen protozoa may explain why no
differences between defaunation and refaunation in either the whole-tract DMD nor
apparent N digestibility occurred in the present study. Zeitz et al. (2012) reported ciliate
protozoa did not affect the digestibility of OM, neutral detergent fibre (NDF) or acid
detergent fibre (ADF), but apparent N digestibility was increased by the presence of
Eudiplodinium maggii by 8%. The lack of differences in N excretion through urine and
body N retention between defaunated and refaunated sheep was in contrast with Santra
et al. (2007a) who reported a lower N urinal excretion and greater N retention in
Chapter 2: Refaunation of sheep after being protozoa-free
57
defaunated sheep. The often increased microbial synthesis in defaunated rumen resulted
in greater N retention (Bird et al. 1994; Jouany 1996), but these were not observed in
the present study. Kreuzer et al. (1986) reported animals consuming diets rich in fibre
increased faecal energy (about 1.26 MJ/day) and urinary energy outputs (about 0.08
MJ/day) than those consuming high starch diets. The urinary energy excretion in the
present study was not lower in defaunated sheep, which is in agreement with Kreuzer et
al. (1986) who fed wethers a diet rich in cellulose content. Reduced energy loss in urine
by defaunation is often reported and related to the reduced NH3 concentration in the
rumen, and the increased MCP supply and greater N retention after defaunation
(Whitelaw et al. 1984; Bird et al. 1994; Jouany 1996). A (non-significant) 15 % higher
MCP supply in defaunated sheep was observed in this study, while N retention and
energy excretion in urine and faeces were not different between treatments. The energy
loss in CH4/GE intake was lower in defaunated than refaunated sheep and these energy
loss fell in the range of 5 to 7% as reported by Hristov et al. (2013).
2.5 Conclusion
These experiments have confirmed that the absence of protozoa from the rumen leads to
an increase in rumen size and has a lower DMP without affecting apparent digestibility.
Longer studies are required to quantify ADG and energetic efficiencies, but the present
studies confirm the efficacy of defaunation as a strategy to reduce enteric CH4
emissions of sheep without adverse effects of digestive consequences.
Chapter 2: Refaunation of sheep after being protozoa-free
58
Higher Degree Research Thesis by Publication
University of New England
Statement of Originality
(To appear at the end of each thesis chapter submitted as an article/paper)
We, the PhD candidate and the candidate’s Principal Supervisor, certify that the
following text, figures and diagrams are the candidate’s original work.
Type of work
Paper numbers
Journal article 31-57
Name of Candidate: Son Hung Nguyen
Name/title of Principal Supervisor: Prof. Roger Stephen Hegarty
__________________ 14 June 2016
Candidate Date
_________________ 14 June 2016
Principal Supervisor Date
Chapter 2: Refaunation of sheep after being protozoa-free
59
Higher Degree Research Thesis by Publication
University of New England
STATEMENT OF AUTHORS’ CONTRIBUTION
(To appear at the end of each thesis chapter submitted as an article/paper)
We, the PhD candidate and the candidate’s Principal Supervisor, certify that all co-authors
have consented to their work being included in the thesis and they have accepted the
candidate’s contribution as indicated in the Statement of Originality.
Author’s Name (please print clearly) % of contribution
Candidate Son Hung Nguyen
80%
Other Authors Graeme Bremner
5%
Margaret Cameron
2%
Roger Stephen Hegarty
13%
Name of Candidate: Son Hung Nguyen
Name/title of Principal Supervisor: Prof. Roger Stephen Hegarty
__________________ 14 June 2016
Candidate Date
_________________ 14 June 2016
Principal Supervisor Date
Chapter 3: Methane production of grazing defaunated sheep
61
Chapter 3
Methane emissions and productivity of defaunated
and refaunated sheep while grazing
S. H. Nguyena, b
, G. Bremnera, R. S. Hegarty
a
a School of Environmental and Rural Science, University of New England, Armidale,
NSW 2351, Australia
b National Institute of Animal Sciences, Hanoi, Vietnam
Paper prepared for submission to Small Ruminant Research
Chapter 3: Methane production of grazing defaunated sheep
62
Abstract
Rumen protozoa produce hydrogen, which is utilised by methanogens to synthesis
enteric methane (CH4) that is a loss of digested energy and has an adverse
environmental impact as a greenhouse gas. The aim of this study was to examine the
effect of the absence of rumen protozoa on pasture intake, ruminal fermentation and
enteric CH4 production of grazing sheep. An incomplete crossover experiment was
conducted with eleven crossbred ewes (6 without [defaunated] and 5 with protozoa
[refaunated]) on 2 × 2 ha pastures with daily CH4 production (DMP) being measured by
GreenFeed Emission Monitoring (GEM) units. It was concluded that grazing defaunated
sheep exhibited lower concentration of rumen ammonia (P = 0.01), but similar
concentrations of total rumen volatile fatty acids compared to refaunated sheep (P >
0.05). The molar proportion of acetate was decreased and butyrate proportion was
increased by defaunation, while the proportion of propionate was unchanged by the
absence of rumen protozoa. Estimated pasture intake was not different between
defaunated and refaunated sheep (P > 0.05) Defaunated sheep tended to have a higher
total dry matter intake (tDMI; P = 0.06), being the sum of pasture intake and pellet
supplement intake. There was a tendency towards a lower CH4 yield (g CH4/kg tDMI; P
= 0.07) in defaunated sheep.
Keywords: Pasture intake, defaunation, methane, sheep
Chapter 3: Methane production of grazing defaunated sheep
63
3.1 Introduction
Recent meta-analysis confirms that removal of ciliate protozoa from the rumen of
ruminants can increase livestock average daily gain by 9% and reduce enteric methane
(CH4) emissions by 11% (Newbold et al. 2015). The positive effect of defaunation on
animal growth is often seen with poor quality roughage diets that are low in nitrogen
content and provide insufficient rumen degradable protein for the growth of rumen
microbes (Bird and Leng 1978; Boodoo et al. 1978; Williams and Coleman 1992). This
may be advantageous in the tropics where forages are often deficient in protein and have
higher fibre content than do temperate grasses (Minson 1990). Since higher fibre
content is associated with a great CH4 yield (Margan et al. 1988), a higher CH4 yield as
well as reduced animal performance can be expected to coincide. This suggests that
elimination of rumen protozoa can improve growth and reduce CH4 emissions of
ruminants grazing on tropical or other low quality pastures. However, there is little data
from grazing animals available to confirm this (Bird and Leng 1984; Hegarty et al.
2000) and no grazing methane data. This study was conducted to quantify whether
effects of defaunation on VFA, DMI, wool growth and CH4 production observed in
controlled feeding studies are also evident in grazing environments.
3.2 Materials and methods
3.2.1 Animals and experimental procedures
The animals and defaunation treatments were described previously in (Chapter 2).
Briefly, twelve crossbred ewes (Border Leicester rams × Merino ewes) about 30 months
of age, were defaunated by treatment with sodium 1-(2-sulfonatooxyethoxy) dodecane
Chapter 3: Methane production of grazing defaunated sheep
64
(Empicol ESB/70AV, Albright and Wilson Australia Ltd, Melbourne) administered at
10 g/day in a 10% v/v solution for three consecutive days. After defaunation one sheep
was slow to recover appetite, so was removed from the study. Eighteen weeks after
defaunation, 5 sheep were re-inoculated with rumen protozoa and were successfully
refaunated for a period of 8 weeks.
Ewes with initial liveweight (± s.e.m) of 56.7 ± 1.9 kg (-P; defaunated) and 57.9 ± 2.0
kg (+P; refaunated) were adapted to the pasture environments in two paddocks adjacent
to each other and adjacent to the paddocks to be used in this experiment. After 2 weeks
of adaptation to pasture, a 56-day grazing study was conducted with the two groups of
sheep managed on 14 day rotation through four paddocks (1 ha paddock, Table 3.1),
with a combination of fixed wire and portable electric fence being used to restrict sheep
movement. There was a 14 day rest period for each paddock before it was re-grazed in
the rotation. The rotation was arranged so that paddock did not bias the estimate of
growth or intake by sheep over the 56 day rotational grazing study. Because two full
rotations were made (each group grazed each paddock twice), analysis of pasture
attributes was analysed for period 1 (rotation 1: d1-28) verses period 2 (rotation 2: day
29-56) and the interaction of period × treatment tested (Table 3.1).
Two GreenFeed Emissions Monitors (GEM) were continuously used to measure CH4
and carbon dioxide (CO2) throughout the 56-day study, with the GEM units moved
during rotation, so sheep had continuous GEM access. Defaunated sheep were always
prevented from physical contact with refaunated sheep or other ruminants by two rows
of electric fencing.
Chapter 3: Methane production of grazing defaunated sheep
65
Table 3.1 Experimental schedule for pasture rotation and data measurements.
Day Activity
Comparative study
-14 Sheep were shorn and adapted to the pasture environment in 2
paddocks adjacent to the experimental paddocks.
-4 Rumen fluid for VFA, ammonia and protozoa were collected,
liveweight and mid-side patches were measured.
0-14
14-28
24-28
28
28-42
Defaunated and refaunated sheep grazed on the experimental
paddocks 1 and 3.
Defaunated and refaunated sheep grazed on the experimental
paddocks 4 and 2
Faecal sampling (period 1)
Rumen fluid for VFA, ammonia and protozoa were collected and
liveweight were measured
Defaunated and refaunated sheep grazed on the experimental
paddocks 3 and 1
42-56 Defaunated and refaunated sheep grazed on the experimental
paddocks 2 and 4
49-53
56
Faecal sampling (period 2)
Rumen fluid for VFA, ammonia and protozoa were collected
liveweight, mid-side patches and whole body fleece weight were
measured
3.2.2 Estimation of pasture green dry matter
Visual assessment of pasture green dry matter on offer (DM; kg/ha) was conducted on
days -4, 17, 31 and 45 using a Crop Circle™ ACS 210 (Holland Scientific, Lincoln NE
USA) sensor coupled to a Trimble ProXRS differential receiver and Ranger data-
logger. The Crop Circle™ sensor emits NIR (880nm) and red (650nm) light and
measures the reflectance coming back from the plant canopy (Lamb et al. 2009). The
values obtained from the device were calibrated to actual on-ground green DM at the
Chapter 3: Methane production of grazing defaunated sheep
66
time of measurement by taking stationary readings of six 30 × 30 cm quadrats. The
Normalised Difference Vegetation Index (NDVI) was calculated from the individual
light reflectance values (NDVI = (NIR(reflectance) - Red(reflectance))/(NIR(reflectance) + Red
reflectance)) and was correlated with total green DM (kg/ha; Trotter et al. 2010). Estimates
of green DM for each NDVI were then calculated using the equation below developed
over the same paddocks (McPhee et al. 2010) with the data being shown in (Figure 3.1).
Green dry matter (kg/ha) = 37.73 × e5.86 × NDVI
(eq 3.1; McPhee et al. 2010).
As the standing dead portion of the sample does not contribute to the Crop Circle™
response, it was not measured by the NDVI, but three quadrat pasture samples in each
paddock were also cut and dried in a fan-force oven at 600 C until constant weight.
These pasture samples were further partitioned into green and dead proportions to
determine their individual biomass with samples of green and dead pastures analysed
for chemical composition using near-infrared spectroscopy (AFIA 2014; Table 3.2).
3.2.3 Predicted dry matter intake and dry matter digestibility
The GrazFeed modelling software (Freer et al. 1997) was used to estimate probable
pasture dry matter intake (pDMI) for sheep in this study based on the quality and
quantity of pasture green and dead DM (Table 3.2) and animal information. GrazFeed
also estimated likely pasture selection by sheep, showing selected dry matter
digestibility (DMD), crude protein (CP) and metabolisable energy (ME) of pastures
(Table 3.3). Total dry matter intake (tDMI) was calculated as the sum of pasture and
pellet DMI. This tDMI was then used to calculate CH4 yield (MY; g CH4/kg tDMI).
Chapter 3: Methane production of grazing defaunated sheep
68
Table 3.2 Pasture green dry matter (GDM), pasture green fraction and chemical analysis of the pastures‡ available to defaunated (-P) and
faunated (+P) sheep rotationally grazing. In each 28 day experimental period, sheep had access to two paddocks and all sheep grazed all
paddocks over the 56-day study.
Treatment Period Paddock number
NDF ADF DMD DOMD OM CP ME Pasture green DM availability (kg/ha)
Pasture dead DM availability (kg/ha)
-P 1 1 61.3 38.6 55.8 53.5 92.2 9.02 7.99 1319 2132
1 4 68.0 42.4 52.1 51.2 96.3 6.63 7.41 1095 2419
2 3 72.9 45.8 46.3 45.1 97.3 5.11 6.04 1350 5154
2 2 72.2 45.5 50.5 48.7 96.4 5.39 6.77 1285 3705
+P 1 3 71.7 44.9 49.8 47.5 96.9 6.51 6.73 1095 3805
1 2 67.1 43.1 52.1 50.9 95.0 6.51 7.33 1001 3087
2 1 64.4 41.1 54.9 54.0 93.6 7.38 7.81 2082 3610
2 4 73.8 46.0 48.5 47.5 96.5 4.32 6.75 1121 3506
s.e.m 2.61 1.66 2.32 2.00 0.49 0.62 0.31 254 1026
Pasture effect 0.01 0.02 0.04 0.02 <0.01 <0.01 0.02 0.11 0.41
Time effect 0.06 0.06 0.16 0.19 0.03 <0.01 0.05 0.08 0.14
Pasture × time effect 0.82 0.87 0.91 0.76 0.49 0.79 0.92 0.54 0.97 ‡Neutral detergent fibre (NDF); acid detergent fibre (ADF); dry matter digestibility (DMD); digestible organic matter in dry matter (DOMD); organic matter (OM); crude
protein (CP); metabolisable energy (ME). All are expressed as % in DM while ME is expressed as MJ/kg DM.
Chapter 3: Methane production of grazing defaunated sheep
69
Table 3.3 Crude protein, dry matter digestibility and metabolisable energy content of
pastures on offer and that estimated to be selected by defaunated (-P) and faunated (+P)
sheep using GrazFeed (Freer et al. 1997).
Parameter
-P (n = 6) +P (n = 5)
Period 1 Period 2 Period 1 Period 2
Description of pasture on offer
Green dry matter (kg/ha) 1207 1318 1048 1602
Dead dry matter (kg/ha) 2276 4429 3446 3558
Dry matter digestibility (DMD; % DM) 52 51 48 52
Crude protein (CP; % DM) 7.8 5.3 6.5 6.0
Metabolisable energy (MJ/kg DM) 7.7 6.4 7.0 7.3
Description of pasture selected (GrazFeed estimate)
Pasture selected DMD (% DM) 77 78 76 77
Pasture selected CP (% DM) 13 14 13 11
Pasture selected ME (MJ/kg DM) 11.1 11.2 10.9 11.2
3.2.4 Methane and carbon dioxide measurement by Greenfeed
Emission Monitoring units
Daily production of methane and CO2 of all sheep was measured by the GEM units
present with each group. A restricted pellet supplement (12 MJ/kg DM and 15% CP,
Table 3.4) with chromium oxide (Cr2O3) inclusion (1.46 mg Cr2O3/kg pellet mix) was
mechanically delivered by the GEM units. Sheep voluntarily placed their heads in the
Chapter 3: Methane production of grazing defaunated sheep
70
GEM shroud and were detected by the radio-frequency identification sensor, triggering
the GEM to progressively release pellets (Hammond et al. 2016). Eructated CH4 and
CO2 were measured while sheep consumed the pellet supplement. Pellets were
dispensed to individual sheep no more frequently than every 4h (total maximum of 5
supplementation events per day). At each supplementation event, up to 5 drops of
pellets were made, with drops being made at 40s intervals and providing 7.5 ± 0.41 g
pellets/drop. This supplementation regime routinely encouraged sheep at the GEM for
CH4 and CO2 measures for at least 2 min while being supplemented.
Table 3.4 Chemical composition of the pellets supplied through the GreenFeed Emision
Monitoring (GEM) unit (g/100g dry matter).
Component GEM Pellets
Dry matter (in feed as-fed) 92.2
Dry matter digestibility 72
Digestible organic matter 71
Inorganic ash 8
Organic matter 92
Neutral detergent fibre 38
Acid detergent fibre 11
Crude protein 15.2
Crude fat 5.5
Metabolisable energy (MJ/kg DM) 12.2
Chapter 3: Methane production of grazing defaunated sheep
71
3.2.5 Analytical procedures
Faecal samples collected from the rectum of all sheep on days 24-28 (period 1) and days
49-53 (period 2), were stored frozen and later dried to constant weight at 600C in a fan-
force oven, ground through a 1-mm screen and concentrations of chromium and silica
(Si in feed and faeces; Barnett et al. 2016) were determined using a portable X-ray
fluorescence spectroscopy (Bruker Tracer III-V pXRF, Bruker Corp, MA USA).
Samples of rumen fluid (20 mL) were collected from each sheep on day 28 and day 56
by oesophageal intubation (using a fresh collection tube used for each animal) for
protozoal enumeration, VFA and NH3 analyses. Rumen pH was measured immediately
using a portable pH meter (Orion 230 Aplus, Thermo scientific, Beverly, MA, USA). A
15 mL subsample was placed in wide-neck McCartney bottle acidified with 0.25 mL of
18 M sulphuric acid and stored at -200C for VFA and NH3 analyses. A 4 mL subsample
was placed in a wide-neck McCartney bottle containing 16 mL of formaldehyde-saline
(4% formalin v/v) for protozoa enumeration. Protozoa were subsequently counted using
a Fuchs–Rosenthal optical counting chamber (0.0625 mm2, 0.2 mm of depth) using a
staining technique adapted from Dehority (1984). The protozoa were differentiated into
large (>100 µm) and small (<100 µm) holotrich and entodiniomorph groupings. The
VFA concentrations were determined by gas chromatography (Nolan et al. 2010) using
a Varian CP 3800 Gas Chromatograph (Varian Inc. Palo Alto, California USA) and
NH3 concentration was analysed by a modified Berthelot reaction using a continuous
flow analyser (San++
, Skalar, Breda, The Netherlands).
Sheep were weighed on days -4, 28 and 56. Clean wool growth rate (CWG), wool yield,
and fibre diameter were determined on the mid-side of the sheep from day -4 to day 56
Chapter 3: Methane production of grazing defaunated sheep
72
by clipping a patch 10 × 10 cm (Oster Golden A5 clippers, blade size 30 model,
Cryogen X, USA). After the wool from the patch was clipped, four sides and one
diagonal were measured and the area of the patch was calculated using Heron’s formula
(De Barbieri et al. 2014). Wool samples were sent to New England Fibre Testing (Pty
Limited, Walcha NSW Australia) to determine yield and fibre diameter. Sheep were
shorn on days -14 and 56 to measure greasy fleece weight.
3.2.6 Statistical analyses
Because both groups of sheep were rotated across all paddocks (twice) over the 56 days,
the role of paddock was accounted for in experimental design and so paddock was not
included in statistical models. However effect of time on pasture growth was assessed
by comparison of means over the first rotation (d1-d28) with the second rotation (d29-
56). Rumen fermentation parameters, DMP, MY, DMI and DMD were analysed using
the repeated-measures analysis of variance using SAS 9.0 (SAS Institute, Cary, NC)
with protozoal treatment, day and protozoa × day interaction as fixed factors. Final
liveweight, ADG, CWG, greasy fleece weight and wool fibre diameter were subject to
analysis of variance. Effects of unbalanced numbers between treatments was not
accounted for. Means were analysed using the least squares means (LSMEANS)
procedure. A probability of error of less than 5% was considered to be statistically
significant.
Chapter 3: Methane production of grazing defaunated sheep
73
3.3 Results
3.3.1 Pastures
The availability of green and dead DM and pasture quality are shown in Table 3.2. The
paddocks did not have a significant difference in green or dead biomass availability (P
> 0.05), but the pasture green DM tended to increase in biomass from period 1 to period
2 (P = 0.08). The chemical composition of pastures in the paddocks was different (P <
0.05) and was affected by period. Pasture NDF and ADF tended to increase (P = 0.06)
between period 1 (67.03% and 42.25%, respectively) and period 2 (70.83% and
44.60%, respectively) while CP and ME significantly decreased (P < 0.05) between
period 1 (7.15% and 7.37 MJ/kg, respectively) and period 2 (5.55% and 6.84MJ/kg,
respectively). Dry matter digestibility and DOMD did not differ between two periods (P
> 0.05).
The chemical composition of selected pasture as estimated by GrazFeed showed a 35%
higher DMD and ME and a 49.8% higher CP than that of the average composition of
pasture biomass available (Table 3.3).
3.3.2 Ruminal fermentation and methane production
Defaunated sheep had a lower rumen pH and concentration of rumen NH3 (P = 0.01)
than did refaunated sheep and the concentration of rumen NH3 was higher in period 2
than period 1 in both groups of sheep (P = 0.01; Table 3.5). The total concentration of
rumen VFA was not affected by either protozoa or period (P > 0.05) but there was a
shift of molar VFA proportion to a reduced proportion of acetate and increased
Chapter 3: Methane production of grazing defaunated sheep
74
proportion of butyrate (P < 0.05) in defaunated sheep while DMP was not different
between defaunated and refaunated sheep (P > 0.05). The MY tended to be lower in
defaunated than refaunated sheep (P = 0.06), with pasture DMI used to calculate MY
being estimated by GrazFeed. The DMP increased between period 1 and period 2 in
both defaunated (7%) and refaunated sheep (11%; P = 0.04) in keeping with greater
pasture biomass availability in period 2. The intake of pellets from GEM units differed
between groups reflecting fewer visits to the GEM and so a lower voluntary intake of
these pellets by the refaunated sheep (Table 3.5).
3.3.3 Dry matter intake, liveweight gain and wool production
Pellet DMI, which was mechanically regulated by the GEM, was higher in defaunated
than refaunated sheep (P < 0.05; Table 3.5). Concentrations of Si and Cr in feed and
faeces were determined, but daily Cr intake of individuals was not constant due to
highly variable consumption of GEM pellets, making it difficult to have a plateau level
in the faeces to estimate pasture intake and Cr data was not used further. Pasture DMI
(pDMI) was, therefore, estimated by GrazFeed and it was estimated to be not affected
by protozoa or period (P > 0.05). Total DMI (tDMI) tended to be greater in defaunated
than refaunated sheep (P = 0.06) due to greater pellet DMI by defaunated than
refaunated sheep (P = 0.03). Estimated DMD was not different between defaunated and
refaunated sheep (P > 0.05) with no difference in Si concentration in faecal DM (Table
3.5). Average daily gain was not affected by protozoa or period (P > 0.05) and there
were no differences in CWG, greasy fleece weight or wool fibre diameter between
defaunated and refaunated sheep in the grazing environment (P > 0.05; Table 3.6).
Chapter 3: Methane production of grazing defaunated sheep
75
Table 3.5 Intake, rumen fermentation parameters and methane emissions of defaunated
(-P) and refaunated sheep (+P) grazing pastures.
Parameter
-P (n = 6) +P (n = 5)
Pooled
s.e
P-value
Period
1
Period
2
Period
1
Period
2
Protozoal
effect
Day
effect
Protozoa
× day
effect
pH
6.38 6.65 6.90 6.54 0.08 0.01 0.60 0.04
NH3-N (mg/L) 33.98 49.46 51.36 62.76 4.87 0.01 0.01 0.68
Total VFA (mM/L) 88.51 92.20 99.58 98.95 8.42 0.91 0.14 0.29
Acetate (molar %) 60.84 61.56 62.93 64.89 1.15 0.03 0.26 0.59
Propionate (molar %) 22.84 22.45 23.90 20.77 0.69 0.62 0.02 0.06
Butyrate (molar %)
14.23 13.83 11.40 11.03 1.14 0.02 0.74 0.99
Acetate/propionate 2.68 2.77 2.64 3.13 0.12 0.19 0.02 0.10
Faecal silica
(mg/kg DM)
5.43 4.77 5.36 3.27 0.92 0.41 0.16 0.45
DMD*
59.51 58.62 55.46 57.32 8.58 0.76 0.95 0.87
DMP (g CH4/day)†
27.52 29.61 27.78 31.21 1.50 0.54 0.04 0.62
pDMI (kg/day) 1.167 1.224 1.170 1.099 0.04 0.12 0.85 0.10
tDMI (kg/day)⁵
1.246 1.306 1.239 1.165 0.03 0.06 0.85 0.08
MY
(g CH4/kg tDMI)⁵
22.09 22.75 22.47 26.72 1.09 0.06 0.04 0.12
Pellet DMI (g/day) 79.6 82.6 68.9 66.2 5.73 0.03 0.98 0.63
Pellet drops/day 11.05 11.47 9.58 9.20 0.79 0.04 0.89 0.62
Total protozoa
(×105 cells/ml)
0 0 15.56 18.21 6.66 0.17
*Dry matter digestibility (DMD) was estimated by silica marker (eq.3.2); † Daily methane production (DMP) was
measured by Greenfeed Emission Monitor; ⁵Total dry matter intake (tDMI; kg/d) was the sum of estimated pasture
DMI by Grazfeed and pellet DMI measured by GEM. Methane yield (MY) was calculated as DMP divided by tDMI.
Chapter 3: Methane production of grazing defaunated sheep
76
Table 3.6 Wool parameters of defaunated (-P) and refaunated sheep (+P) grazing
pastures.
Parameter
Protozoal treatment Difference (-P) - (+P)
with pooled s.d P-value
-P (n=6) +P (n=5)
Final liveweight (kg) 68.32 68.72 -0.4 ± 1.04 0.93
Average daily gain (g/day) 187.5 181.2 +6.3 ± 16.2 0.79
Clean wool growth (μg/cm3/d) 724.3 747.2 -22.9 ± 119 0.77
Greasy fleece weight (kg) 1.30 1.27 +0.03 ± 0.21 0.54
Wool fibre diameter (μm) 30.42 29.68 +0.74 ± 3.42 0.75
3.4 Discussion
3.4.1 Animal productivity
While pasture DMI and DMD were not measured directly in this study; pDMI was
estimated by GrezFeed and DMD by Si concentration in pasture and faecal DM and no
protozoa effects on these were observed. Lower DMI and DMD in defaunated
ruminants are often reported in the literature (Newbold et al. 2015), probably due to the
loss of protozoal fibrolytic activity and longer particle retention of ruminal digesta
associated with the slower rumen outflow and greater rumen fill associated with lower
ruminal DMD (Eugène et al. 2004a). The lack of differences in DMI and DMD due to
protozoa indicates that the only scope for protozoa to affect animal performance in this
study would have been through changing the nutrients array or quantity from the
fermentation and microbial growth processes. Considering the lower rumen NH3
concentration in defaunated sheep, it is likely that defaunated sheep did have lower
Chapter 3: Methane production of grazing defaunated sheep
77
rumen proteolysis as defaunation often increases microbial protein outflow (Newbold et
al. 2015). It was unexpected that defaunated sheep did not have greater wool growth
response than refaunated sheep consuming pasture as wool growth is responsive to
amino acid supply (Reis et al. 1973). Certainly, wool growth of crossbred ewes used in
this study is less than from Merino sheep which have been often shown responses
following defaunation (Bird and Leng 1984), and this low wool growth potential may
be why a wool growth response of defaunated sheep in the current study was not
apparent.
The lack of effect of protozoa on ADG and wool production on this low protein pasture
is in contrast to the literature. Bird and Leng (1984) observed a 23% greater rate of
ADG and a 19% greater wool growth rate in defaunated compared to faunated lambs
grazed on a green oats forage. Protozoa-free lambs born from defaunated ewes were
also 4-8% heavier than were lambs born from faunated ewes and wool growth was
greater in protozoa-free lambs grazed on fescue dominant pastures (Hegarty et al.
2000). Sheep in the current study, though more mature than those of Bird and Leng
(1984), were still having a high protein demand for growth to support an ADG of
almost 200 g/day. Hence, it is again surprising that defaunation did not cause a
significant difference in ADG. The concentration of NH3 in the rumen of defaunated
sheep was below 50 mg/L which is below microbial protein yield required for the
optimal growth of rumen bacteria (Satter and Slyter 1974). However, both microbial
protein outflow and dietary protein intake by sheep need to be considered in explaining
why ADG and wool growth following defaunation was not increased by defaunation as
expected in the grazing environment.
Chapter 3: Methane production of grazing defaunated sheep
78
3.4.2 Rumen fermentation and daily methane production
The reduced rumen pH in defaunated sheep confirms that rumen protozoa may have a
role in stabilising rumen pH and avoiding the risk of acidosis associated with sudden
fall in pH (Jouany and Ushida 1999), but this is not often seen (Newbold et al. 2015)
and acidosis would have be unlikely on the fibrous pastures. The reduced concentration
of rumen NH3 following defaunation, however, is consistent between the current study
and in the literature (Newbold et al. 2015). This is due to the reduced deamination of
bacterial protein through protozoa predation and less feed-protein being degraded in the
rumen of the absence of protozoa (Williams and Coleman 1992; Jouany and Ushida
1999).
A reduced concentration of total VFA following defaunation is often reported (Newbold
et al. 2015) being ascribed to (i) reduced ruminal DM fermentation (Eugène et al.
2004a; Newbold et al. 2015) (ii) increased fermented materials being captured in rumen
microbial cells rather than producing VFA for the host (Williams and Coleman 1992)
and (iii) a larger rumen volume, but reduced total VFA concentration was not induced
by defaunation in the current study. This is consistent with no difference in faecal Si,
suggesting neither ruminal nor whole tract digestibility were affected by protozoa. The
reduced proportion of acetate following defaunation is consistent with Eugène et al.
(2004a). Differences in VFA proportions after defaunation are often seen as an
increased proportion of propionate and a decreased proportion of acetate (Eugène et al.
2004a) due to removing rumen protozoa which synthesise acetic acid rather than
propionic acid (Williams and Coleman 1992), therefore reducing hydrogen availability
Chapter 3: Methane production of grazing defaunated sheep
79
to methanogens and the amount of CH4 produced. The butyrate content was unusually
high, and the higher proportion of butyrate in the rumen VFA from defaunated sheep
relative to refaunated sheep in the current study is inconsistent with Williams and
Coleman (1992) or Newbold et al. (2015).
The lack of effect on DMP due to defaunation in this study is inconsistent with housed
studies where defaunation is often associated with average 11 to 13% CH4 mitigation
(Hegarty 1999; Newbold et al. 2015). However, the mechanism through which DMP is
reduced by defaunation is not well understood (Newbold et al. 2015). Defaunation
decreases methanogens existing as endo and ecto-symbionts with rumen protozoa
(Finlay et al. 1994; Tokura et al. 1997; Kumar et al. 2013), but CH4 emissions are not
always decreased (Kumar et al. 2013). This may be due to the complexity of multiple
rumen microbes being involved following defaunation (Morgavi et al. 2010). Hence,
reduced CH4 emissions following defaunation is not always a consistent consequence of
the decreased symbiotic habitat of methanogens (Morgavi et al. 2012). The loss of the
cilate-associated methanogens in defaunated animals may have been compensated for
by an increase in population of cellulolytic ruminococci which are large hydrogen
producers (Mosoni et al. 2011). The changes in the methanogenic population following
defaunation are inconsistent among studies (McAllister and Newbold 2008; Mosoni et
al. 2011; Morgavi et al. 2012; Kumar et al. 2013). Therefore, our understanding in the
relationship between rumen ciliate-associated methanogens and CH4 production in
defaunated animals is still limited.
Chapter 3: Methane production of grazing defaunated sheep
80
3.5 Conclusion
This study reported for the first time that MY tended to be lower in protozoa-free sheep
relative to refaunated sheep in the grazing environment, though it is acknowledged that
pasture intake used in calculating MY was estimated from modelling. An accurate
measure of pasture intake, therefore, is required to confirm this finding. The lack of
animal performance (ADG and CWG) responses to defaunation, together with lack of
effect on DMP suggest the need for caution in assuming defaunation will improve the
productivity and reduce the environmental impact of grazing ruminants.
Chapter 3: Methane production of grazing defaunated sheep
81
Higher Degree Research Thesis by Publication
University of New England
Statement of Originality
(To appear at the end of each thesis chapter submitted as an article/paper)
We, the PhD candidate and the candidate’s Principal Supervisor, certify that the
following text, figures and diagrams are the candidate’s original work.
Type of work
Paper numbers
Journal article 61-80
Name of Candidate: Son Hung Nguyen
Name/title of Principal Supervisor: Prof. Roger Stephen Hegarty
__________________ 14 June 2016
Candidate Date
_________________ 14 June 2016
Principal Supervisor Date
Chapter 3: Methane production of grazing defaunated sheep
82
Higher Degree Research Thesis by Publication
University of New England
STATEMENT OF AUTHORS’ CONTRIBUTION
(To appear at the end of each thesis chapter submitted as an article/paper)
We, the PhD candidate and the candidate’s Principal Supervisor, certify that all co-authors
have consented to their work being included in the thesis and they have accepted the
candidate’s contribution as indicated in the Statement of Originality.
Author’s Name (please print clearly) % of contribution
Candidate Son Hung Nguyen
75%
Other Authors Graeme Bremner
15%
Roger Stephen Hegarty
10%
Name of Candidate: Son Hung Nguyen
Name/title of Principal Supervisor: Prof. Roger Stephen Hegarty
__________________ 14 June 2016
Candidate Date
_________________ 14 June 2016
Principal Supervisor Date
Chapter 4: Nitrate and defaunation affect lamb productivity
83
Chapter 4
Use of dietary nitrate to increase productivity and
reduce methane production of defaunated and
faunated lambs consuming protein deficient chaff
S. H. Nguyena, c
, M. C. Barnetta, b
, R. S. Hegartya
a School of Environmental and Rural Science, University of New England, Armidale,
NSW 2351, Australia
b School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga,
NSW 2678, Australia
c National Institute of Animal Sciences, Hanoi, Vietnam
Animal Production Science, 2016, 56. 290-297
Chapter 4: Nitrate and defaunation affect lamb productivity
84
Abstract
The effects of dietary nitrate supplementation and defaunation on methane (CH4)
emissions, microbial protein outflow, digesta kinetics and average daily gain were
studied in lambs fed chaff containing 4.1% crude protein in dry matter. Twenty ewe
lambs were randomly allocated in a 2×2 factorial experiment (0 or 2% nitrate
supplementation and defaunated or faunated protozoal state). Nitrate supplementation
increased blood methaemoglobin concentration (P < 0.05), rumen volatile fatty acid and
ammonia concentrations, dry matter intake, microbial protein outflow, average daily
gain, dry matter digestibility, clean wool growth and wool fibre diameter (P < 0.01).
Nitrate increased CH4 production (g/d) due to greater dry matter intake, but did not
affect CH4 yield (g CH4/kg dry matter intake). Nitrate-supplemented lambs had a
shorter total mean retention time of digesta in the gut (P < 0.05). Defaunation reduced
CH4 production and CH4 yield by 43 and 47%, but did not cause changes in dry matter
intake, microbial protein outflow, average daily gain or clean wool growth. Defaunation
decreased total volatile fatty acids and the molar percentage of propionate, but increased
the molar percentage of acetate (P < 0.05). Interactions were observed such that
combined treatments of defaunation and nitrate supplementation increased blood
methaemoglobin (P = 0.04), and decreased CH4 yield (P = 0.01) relative to other
treatment combinations.
Keywords: Methanogensis, protozoa, methane, sheep
Chapter 4: Defaunation and nitrate effects on lamb productivity
85
4.1 Introduction
Residues of crops and agricultural by-products are the major feed sources for livestock
in tropical and subtropical regions, but they are often of low to moderate digestibility
with low levels of protein and minerals (Preston 1995). Ruminant production from these
low-quality feeds is limited by the deficiency of absorbed amino acids and energy,
resulting from decreased feed intake and digestibility due to slow growth and
fermentation by rumen microbes (Leng 1990).
Removal of protozoa from the rumen (defaunation) increases average daily gain by 11%
(Eugène et al. 2004a) due to increased bacterial biomass and increased availability of
protein at the duodenum (Bird and Leng 1978; Jouany 1996). Defaunated cattle grew at
a 43% greater rate than faunated cattle on the same intake of a low-protein molasses
based diet (Bird and Leng 1978) while defaunated lambs showed increased growth rate
and wool growth on a low-protein diet (Bird et al. 1979). Defaunation also decreases
enteric methane (CH4) production (Hegarty 1999; Newbold et al. 2015) by eliminating
methanogens that exist as endo- and ecto-symbionts with ciliate protozoa (Finlay et al.
1994) and by often changing the molar proportions of volatile fatty acids to a greater
proportion of propionate and less proportion of butyrate (Eugène et al. 2004a). In
constrast, studies of isolated lambs raised without protozoa from birth and defaunated
ewes had been shown that absence of rumen protozoa did not reduce CH4 production
(Bird et al. 2008; Hegarty et al. 2008). Therefore, there is a lack of certainty whether
CH4 emissions are decreased by defaunation.
Chapter 4: Defaunation and nitrate effects on lamb productivity
86
Dietary nitrate has shown potential to decrease CH4 emissions from ruminants with a
consistent and persistent efficacy (Guo et al. 2009; Nolan et al. 2010; van Zijderveld et
al. 2010; van Zijderveld et al. 2011; Lee and Beauchemin 2014). This is because
hydrogen (H2) is used by microbes to reduce carbon dioxide (CO2) to CH4 (Nolan
1999), but when nitrate is present in the rumen, approximately 2 moles of H2 will be
needed to convert one mole of nitrate to nitrite and 6 moles of H2 will be required in
order to reduce this nitrite to ammonia (Allison and Reddy 1984). A review by Leng
and Preston (2010) concluded that the use of nitrate as a H2 sink could theoretically
reduce CH4 production by 16-50%, depending on diet and the inclusion rate of nitrate,
but there is little data on productivity of nitrate-supplemented ruminants on protein
deficient roughage. Previous studies have largely focused on a comparison of nitrate
and urea as non-protein nitrogen (NPN) sources for ruminant diets and on reducing CH4
production (Nolan et al. 2010; Li et al. 2012; de Raphélis-Soissan et al. 2014). Despite
these potential benefits of nitrate supplements, little is known about the effects of nitrate
on microbial fermentation and growth in the rumen without protozoa, especially in
animals offered diets unbalanced for nitrogen and energy. This experiment aimed to
quantify the effects of nitrate as a source of NPN and the interaction with defaunation
on CH4 production and productivity of lambs offered a protein deficient chaff diet.
4.2 Materials and methods
4.2.1 Animals and feeding
All protocols for care and treatment of the sheep were approved by the University of
New England Animal Ethics Committee (AEC 14-083). Merino ewe lambs (n = 20; 38
Chapter 4: Defaunation and nitrate effects on lamb productivity
87
± 1.9 kg; 13 months of age) were selected and acclimated to a diet of oaten chaff.
Lambs were allocated to dietary nitrogen (N) levels by stratified randomisation based on
liveweight. The experiment was a 2×2 factorial design (calcium nitrate supplementation
at 0 or 3.1%; protozoa status either defaunated or faunated). The diet of 3.1% calcium
nitrate (~2% NO3 as 5Ca(NO3)2.NH4NO3.10H2O, Bolifor CNF, Yara, Oslo, Norway)
was prepared by sprinkling a dilute solution of the nitrate onto oaten chaff while the
chaff was tossed in a rotary feed mixer (+NO3 ; Table 4.1). Another diet (control) was
only oaten chaff (-NO3; Table 4.1).
The experiment lasted for 93 days. Lambs were gradually adapted to nitrate-
supplemented oaten chaff from day 0 to day 15 from an initial inclusion of NO3 of 1%
up to 2%, with the dose of calcium NO3 increased every two days. After this period of
NO3 adaptation, lambs were given ad libitum access to nitrate-supplemented oaten chaff
with 2% NO3 from day 16 to day 40. Lambs were placed on restricted intake (80%
individual ad libitum intake) 5 days before entering respiration chambers on day 45 to
day 50 and continued receiving restricted intake from day 50 to day 59 for study of
digesta kinetics and total excreta collection and from day 59 to day 64 for repeated
measure of CH4 emissions in respiration chambers. Lambs resumed ad libitum intake on
day 64 until the end of the experiment. Lambs were fed twice daily in two equal
portions at 0930 and 1500 hours. Water was available at all times.
Chapter 4: Defaunation and nitrate effects on lamb productivity
88
Table 4.1 Chemical composition of the oaten chaff and nitrate-supplemented chaff
(g/100g dry matter).
Component Oaten chaff
(-NO3)
Oaten chaff (+NO3)
(3.1% Bolifor)
Dry matter (g/100g as fed) 90.2 89.6
Dry matter digestibility 71 70
Digestible organic matter 67 66
Inorganic ash 6.4 7.3
Organic matter 93.6 92.7
Neutral detergent fibre 49 48
Acid detergent fibre 26 25
Crude protein 4.1 7.1
Metabolisable energy (MJ/kg) 10.6 10.4
Nitrate-nitrogen (mg/kg) 60.3 4,300
Nitrate 0.03 1.9
Bolifor CNF: 5Ca(NO3)2.NH4NO3.10H2O (63.12% nitrate in Bolifor CNF)
4.2.2 Feed sampling and chemical analyses
Samples of oaten chaff (100 g) were collected before and after each mix of feed and
stored in -200 C. All samples were pooled and sub-samples were taken to analyse for
chemical composition (Table 4.1). Feed samples were analysed by the NSW DPI Feed
Quality Service, Wagga Wagga Agriculture Institute, NSW, Australia. Feed dry matter,
crude protein, acid detergent fibre, neutral detergent fibre, and inorganic ash were
determined by wet chemistry. Feed dry matter digestibility and digestible organic matter
were determined by near-infrared spectroscopy (AFIA 2014).
Chapter 4: Defaunation and nitrate effects on lamb productivity
89
4.2.3 Defaunation of lambs
Ten lambs were offered lucerne cereal mix supplemented with coconut oil distillate
(COD) with initial inclusion of 3% to 5% of COD over 7 days to supress rumen
protozoa. After 7 days feeding COD, lambs were fasted for 24h and orally dosed with
sodium 1-(2-sulfonatooxyethoxy) dodecane (Empicol ESB/70AV, Albright and Wilson
Australia Ltd, Melbourne) administered at 10 g/d in a 10% v/v solution for three
consecutive days to remove protozoa. Feed was withheld during this period. Animals
required 12 days to recover to their pre-treatment voluntary intake and the three day
dosing with Empicol was then repeated. Defaunated lambs were offered lucerne cereal
mix during second drenching period and a further 14 days after the second drenching
program to recover from defaunation treatment. Fourteen days after second drenching,
defaunated and faunated lambs were given ad libitum access to oaten chaff for 7 days
before day 0. During the defaunation period, the 10 faunated lambs were restricted fed
at their maintenance requirement (CSIRO 2007) to prevent divergence in liveweight
while the defaunated group was being prepared.
4.2.4 Blood methaemoglobin
Blood was sampled between 2.5 and 3 h after morning feeding on days 0, 15, 50 and 85.
A sample of 8 mL was taken from a jugular vein, using lithium heparinised vacutainers
(BD Franklin Lakes, NJ, USA). Whole blood methaemoglobin (MetHb) concentration
was determined within 30 min using a blood gas analyser (ABL 800 Flex, Radiometer,
BrØnshØj, Denmark).
Chapter 4: Defaunation and nitrate effects on lamb productivity
90
4.2.5 Methane production
Daily CH4 production (DMP; g CH4/day) of each lamb was measured in open-circuit
respiration chambers over 2×22-h consecutive periods (Bird et al. 2008). Lambs were
placed in individual respiration chambers by 1100 hours, with their feed and water
available inside the chambers. The chambers were opened to collect feed refusals, clean
faecal trays, and supply fresh feed and water at 0900 hours the following day and then
were resealed at 1100 hours.
Sub-samples of air within each chamber and of the ambient air were collected every 13
min into Tedlar gas sampling bags (Supelco, Bellefonte, PA, USA) continuously over
the 22 h of confinement for analysis. Methane concentration was measured by a
photoacoustic gas analyser (Innova Model 1312, AirTech Instruments, Ballerup,
Denmark). Recovery of CH4 through the chambers was determined by injection of a
known volume of CH4 and measurement of CH4 concentration every 2 min for 20 min,
with recovery of the dose being calculated by integrating the area under the
concentration curve over time.
4.2.6 Digestibility, digesta kinetics and microbial protein outflow
Lambs were placed in metabolism cages and a 6-day collection of faecal and urinary
output was conducted. All faecal output over the 6 days was collected and weighed with
feed DMI and faecal DM output used to determine dry matter digestibility (DMD).
Concurrent with determining DMD, the mean retention time (MRT) of digesta was
estimated in all lambs over 6 days by reference to faecal excretion of a dosed particle-
Chapter 4: Defaunation and nitrate effects on lamb productivity
91
phase marker (5 g per lamb of Cr-mordanted NDF from oaten chaff) and liquid-phase
marker (5 g per lamb of CoEDTA from AVA Chemicals Pty Ltd. Mumbai, India in
45mL of Milli-Q water). The non-digestible Cr-mordanted NDF was prepared in
accordance with Udén et al. (1980) and with CoEDTA administered via intubation
directly into the rumen as a single dose at 10:00 hours on day 53. Faecal samples were
collected every 2 h for the first 24 h, starting 8 h after marker administration, then every
4 h for the next 48 h, every 8 h for the next 24 h, and every 12 h for the next 48 h.
Dry matter content of feed and faeces were determined by drying samples at 60°C in a
fan-forced oven to a constant weight. Samples were ground through a 1 mm sieve
before analysis of Cr and Co concentrations (Barnett et al. 2016) using a portable X-ray
fluorescence spectrometer (Bruker Tracer III-V pXRF, Bruker Corp, Billerica, MA
USA). Analysis of digesta kinetics was undertaken using non-linear curve fitting
algorithms of WinSAAM (Aharoni et al. 1999).
The concentration of allantoin in the urine was determined by the colourimetric method
(IAEA 1997), using a UV-1201 spectrophotometer (Shimadzu, Japan) reading at
522nm. The yield of total microbial crude protein (MCP) from the rumen was
calculated from allantoin excretion using equations of Chen et al. (1992).
Chapter 4: Defaunation and nitrate effects on lamb productivity
92
4.2.7 Rumen fluid sampling, ammonia, volatile fatty acid
concentrations, and protozoal enumeration
Samples of rumen fluid (20 mL) were collected from each lamb before feeding using
oesophageal intubation for protozoal enumeration, volatile fatty acid (VFA) and
ammonia (NH3) analyses on days 0, 25, 39, 65 and 92. Rumen pH was measured
immediately using a portable pH meter (Orion 230 Aplus, Thermo scientific, Beverly,
MA, USA). A 15 mL subsample was placed in wide-neck McCartney bottle acidified
with 0.25 mL of 18 M sulphuric acid and stored at -200C for VFA and NH3 analyses. A
4 mL subsample was placed in wide-neck McCartney bottle containing 16 mL of
formaldehyde-saline (4% formalin v/v) for protozoa enumeration. Protozoa were
counted using a Fuchs–Rosenthal optic counting chamber (0.0625 mm2 and 0.2 mm of
depth) using a staining technique adapted from the procedure described by Dehority
(1984). The protozoa were differentiated into large (>100 µm) and small (<100 µm)
holotrichs and entodiniomorphs. The VFA concentrations were determined by gas
chromatograph (Nolan et al. 2010) using a Varian CP 3800 Gas Chromatograph (Varian
Inc. Palo Alto, California USA) and NH3 concentration was analysed using a modified
Berthelot reaction using a continuous flow analyser (San++, Skalar, Breda, The
Netherlands).
4.2.8 Liveweight and clean wool growth
Lambs were weighed in the morning prior to feeding on days 0, 15, 21, 30, 65 and 93 to
monitor liveweight and determine average daily gain (ADG) over the experiment. Clean
wool growth (CWG) rate, wool yield, and fibre diameter were determined on the mid-
Chapter 4: Defaunation and nitrate effects on lamb productivity
93
side of the sheep from day 25 to day 92 by clipping a patch 10×10 cm (Oster Golden A5
clippers, blade size 30 model, Cryogen X, USA). After the wool from the patch was
clipped, four sides and one diagonal were measured and the area of the patch was
calculated using Heron’s formula (De Barbieri et al. 2014). Wool samples were sent to
New England Fibre Testing Pty Limited to determine yield and fibre diameter.
4.2.9 Statistical analyses
Data was statistically analysed using SAS 9.0 (SAS Institute, Cary, NC). Data for
rumen fermentation characteristics, digesta kinetics, CWG, MCP outflow and DMP
were subject to analysis of variance in PROC GLM; factors being protozoa, NO3 and
protozoa × NO3 interaction. For analysis of final liveweight and ADG, the model used
the initial liveweight as a covariate. For parameters which had more than one measures,
all measures were averaged. Homogeneity of variance and normal distribution were
tested using PROC UNIVARIATE before statistical analysis. Data on MetHb and
protozoa count were log-transformed before statistical analysis. For all analyses, means
were analysed using the least squares means (LSMEANS) procedure and a probability
of < 5% was considered to be statistically significant.
4.3 Results
4.3.1 Blood methaemoglobin concentration
The averaged, blood MetHb concentration over the whole experiment was significantly
increased by defaunation (P = 0.03) and by supplementation of NO3 (P < 0.01) and
there was a significant interaction between defaunation and NO3 supplementation on
Chapter 4: Defaunation and nitrate effects on lamb productivity
94
MetHb (P = 0.04; Table 4.2). In defaunated lambs on NO3, two lambs were observed
having MetHb values of 18.1 and 18.3% on day 50 and one lamb observed with MetHb
value of 19.1% on day 85. In faunated lambs on NO3, the highest MetHb was one lamb
found with 6.2% of MetHb on day 50.
Table 4.2 Rumen fermentation characteristics, concentration of methaemaglobin
(MetHb) and protozoal population of defaunated (-P) and faunated lambs (+P) fed diets
of oaten chaff with or without nitrate (NO3) supplementation.
Parameter
Treatment
Pooled
s.e.
P-Values
-P
+P
P NO3 P ×
NO3 - NO3
(n = 5)
+ NO3
(n = 5)
- NO3
(n = 5)
+ NO3
(n = 5)
Rumen pH 6.72
6.61
6.69
6.57
0.03 0.16 <.001 0.98
Total VFA (mM/L) 32.71
37.71
35.17
49.03
3.10 0.04 0.01 0.17
Acetate (mol %) 72.89
72.62
68.15
71.02
0.95 <.01 0.18 0.11
Propionate (mol %) 17.15
15.42
20.63
17.79
1.18 0.02 0.06 0.63
Butyrate (mol %) 8.00
9.58
9.51
9.42
0.75 0.38 0.34 0.28
Acetate : propionate
ratio 4.37
4.76
3.34
4.07
0.30 0.01 0.08 0.59
NH3-N (mg/L) 6.04
21.51
11.21
30.70
2.72 0.02 <.001 0.46
MetHb (%)
0.91 5.48 0.87 1.55 0.94
0.03
<0.01
0.04
Total protozoa
(×105/mL)
0 0 4.78
6.51
0.61 0.20
Small
entodiniomorph 0 0 4.08 5.54 0.53 0.19
Large
entodiniomorph 0 0 0.18 0.13 0.08 0.51
Small holotrich 0 0 0.50 0.79 0.19 0.33
Large holotrich 0 0 0.02 0.05 0.02 0.42
Chapter 4: Defaunation and nitrate effects on lamb productivity
95
4.3.2 Rumen fermentation and methane emissions
Defaunated lambs remained protozoa-free throughout the experiment. In faunated
lambs, NO3 supplementation did not affect protozoal population (P > 0.05; Table 4.2).
Small entodiniomorphs in both NO3 and non-nitrate supplemented lambs accounted for
85% of total protozoa.
Nitrate supplementation significantly increased concentration of NH3-N and total VFA
(P < 0.05; Table 4.2). There was a tendency towards a lower molar percentage of
propionate (P = 0.06) and higher molar ratio of acetate to propionate (P = 0.08) in
nitrate-supplemented lambs. Defaunation, contrastingly, decreased total VFA, NH3-N
concentration and molar percentage of propionate. Defaunation increased the molar
percentage of acetate and molar ratio of acetate to propionate (P < 0.05). Rumen pH
was not affected by defaunation, but was decreased by NO3 supplementation (Table
4.2). No interactions between defaunation and NO3 were found for any rumen
fermentation parameter.
Daily methane production was significantly decreased by defaunation, but was
increased by NO3 supplementation (P < 0.001; Table 4.3) with no interaction between
defaunation and NO3 for DMP. There was a positive correlation between DMI and
DMP (DMP = 0.014 DMI - 3.38; r2
= 0.75, P = 0.001), such that DMP was significantly
increased by higher DMI. Defaunation significantly decreased CH4 yield (MY, g
CH4/kg DMI) but NO3 did not change MY. However, there was a significant interaction
between defaunation and NO3 (P = 0.01) in MY such that in defaunated lambs, NO3 did
not change MY, but in faunated lambs, NO3 decreased MY by 25%. Nitrate-
Chapter 4: Defaunation and nitrate effects on lamb productivity
96
supplemented defaunated lambs had lower MY than nitrate-supplemented faunated
lambs (P < 0.001; 10.14 v. 14.20 g/kg DMI).
4.3.3 Performances and digestion
Productivity of lambs was significantly increased by NO3 supplementation, but was not
affected by defaunation. Supplementation of oaten chaff with NO3 significantly
increased DMI from 622 to 895 g/d and DMD from 57.8 to 64.5% (P < 0.001; Table
4.3). Nitrate supplementation significantly increased MCP outflow, ADG, CWG and
wool fibre diameter (P < 0.01). The intakes of ME and CP were significantly increased
by NO3 supplementation from 3.8 to 6.1 MJ/d and from 14.9 to 41.7 g/d, respectively.
Defaunation did not change DMI, ADG, MCP outflow or CWG, but decreased DMD (P
= 0.05). There were no significant interactions between defaunation and NO3
supplementation for productivity parameters.
Digesta kinetics as characterised by MRT of both rumen solute and particulate fractions
was significantly affected by both defaunation and NO3 supplementation. Defaunation
significantly increased rumen MRT of solute and particulate fractions (P < 0.05) while
NO3 supplementation significantly decreased rumen MRT of these fractions (P < 0.05).
There was a negative correlation between MRT and DMI across all data (particulate
MRT = 70.8 - 0.028 DMI, r2 = -0.52, P = 0.038 and solute MRT = 52.2 - 0.028 DMI, r
2
= -0.67, P = 0.004), such that greater DMI was associated with shorter marker MRT in
the rumen. Despite the slowing effect of defaunation and accelerating effect of NO3 on
rumen MRT, there were no significant effects of defaunation or NO3 on MRT of solute
Chapter 4: Defaunation and nitrate effects on lamb productivity
97
and particulate fractions in the hindgut. There were no interactions between defaunation
and NO3 in other gut segment, except hindgut particulate MRT (P = 0.02).
Table 4.3 Intake, productivity, methane emissions and digesta kinetics of defaunated (-
P) and faunated lambs (+P) fed diets of low-protein oaten chaff with or without nitrate
(NO3) supplementation.
Parameter
Treatment
Pooled
s.e.
P-Values
-P
+P
P NO3 P ×
NO3 - NO3
(n = 5)
+ NO3
(n = 5)
- NO3
(n= 5)
+ NO3
(n= 5)
DMI (g/d) 620.71
849.32
624.61
941.72
53.79 0.37 <.001 0.41
ME intake (MJ/d) 3.61 5.75 4.08 6.48 0.45 0.20 <.001 0.77
CP intake (g/d) 13.97 39.23 15.77 44.21 2.75 0.24 <.001 0.57
Final LW(kg) 35.42
40.31
32.62
39.84
1.30 0.14 <.001 0.37
ADG (g/d) -0.95
54.55
-32.81
49.26
14.77 0.21 <.001 0.39
CWG (µg/cm2.d)
468
662
482
704
53.5 0.61 <.01 0.79
Wool fibre diameter
(µm) 18.85
22.87
19.58
21.96
0.82 0.96 <.01 0.35
DMP (g CH4/d) 3.49 7.65 7.13 12.29 0.85 <.001 <.001 0.57
MY (g CH4/kg DMI)* 7.30
10.14
18.88
14.20
1.31 <.001 0.50 0.01
DMD (%) 54.88
63.77
60.81
65.25
1.72 0.05 <.01 0.22
MCP outflow (g/d) 3.17
8.55
3.14
8.29
1.45 0.89 <.01 0.96
Rumen particulate
MRT(h) 40.20
32.83
29.10
21.68
1.74 <.001 <.01 0.98
Hindgut particulate
MRT(h) 15.05
20.53
21.97
14.70
2.33 0.82 0.71 0.02
Total particulate MRT(h) 55.25
53.35
50.82
36.37
3.66 0.01 0.05 0.11
Rumen solute MRT(h) 25.37
19.20
20.75
14.10
1.90 0.03 0.01 0.90
Hindgut solute MRT(h) 12.25 12.65 15.20 10.28 1.52 0.85 0.16 0.11
Total solute MRT(h) 37.63
31.85
35.95
24.38
3.22 0.18 0.02 0.38
(n = 4) during measures of methane emissions and total collection; Dry matter intake (DMI); Average daily gain
(ADG); Clean wool growth (CWG); Daily methane production (DMP); Methane yield (DMD); Dry matter
digestibility (DMD); Microbial crude protein (MCP); Mean retention time (MRT); *DMI was calculated during
restricted intake period.
Chapter 4: Defaunation and nitrate effects on lamb productivity
98
4.4 Discussion
4.4.1 Effects of nitrate on blood MetHb concentration and protozoa
population
Nitrate toxicity remains a major constraint to commercial NO3 feeding because
excessive NO3 in the rumen may accumulate nitrite (NO2) concentrations in the rumen
and then blood. Nitrite in blood reduces the ferric ion of haemoglobin and transforms
the molecule to MetHb (Lundberg et al. 2008), which is unable to transport oxygen to
tissues. Methaemoglobinaemia is diagnosed if more than 30% of haemoglobin is
present on MetHb (Bruning-Fann and Kaneene 1993). In this study, feeding faunated
lambs with 3.1% calcium NO3 (~2% NO3) maintained low MetHb concentration in
agreement with previous studies when NO3 was supplemented at levels up to 2.6% by
gradually introducing NO3 to allow adaptation of rumen microbes (Nolan et al. 2010;
van Zijderveld et al. 2010; Li et al. 2012). This is because rumen microbes are capable
of reducing NO3 or NO2 to NH3 as NO3 is introduced as reviewed by Leng and Preston
(2010).
Defaunated lambs in the present study showed increased MetHb concentration after 85
days of feeding NO3, suggesting that protozoa may have an important role in the
reduction of NO3 or NO2 in the rumen and consequently formation of MetHb in the
blood of sheep. Lin et al. (2011) incubated different microbial fractions of whole rumen
fluid, protozoa, bacteria, and fungi to assess their ability to reduce NO3. The authors
found that NO3 disappearance rate was similar in whole rumen fluid and protozoal
fractions. Nakamura and Yoshida (1991) also reported that NO3 and NO2 disappearance
Chapter 4: Defaunation and nitrate effects on lamb productivity
99
rates in the rumen of faunated sheep were faster than in defaunated sheep and lower
MetHb was observed in faunated sheep, potentially indicating active involvement of
protozoa in the reduction of NO3 and NO2. However, there were no clinical signs of
NO3 toxicity in either defaunated and faunated lambs when 2% NO3 was supplemented
in these ad libitum fed lambs.
The protozoal population was not affected by NO3 supplementation in this study; this
agreed with previous studies (Nolan et al. 2010; van Zijderveld et al. 2010; Li et al.
2012).
4.4.2 Effects of nitrate supplementation and defaunation on
performances and digestion
Oaten chaff used in this study as a basal diet for lambs was characterized by a low
protein content (41 g CP per kg DM) providing only 4 g of CP per MJ of ME.
Consequently, this diet was inadequate to support maintenance of growing lambs
(CSIRO 2007), resulting in losing weight in lambs without NO3 supplementation.
Similarly, the average value of 7 g of CP per MJ of ME in the nitrate-supplemented diet
was below that required to support rumen fermentation required for growing sheep
(CSIRO 2007). The averaged concentrations of NH3-N in the rumen increased from 8.6
to 26 mg/L rumen fluid with NO3 and were associated with increased MCP outflow
(3.2 to 8.4 g/d). However, it was suggested by Satter and Slyter (1974) that 20-50 mg
NH3-N/L rumen fluid is required to maintain growth of rumen bacteria with forage
diets, so the amount of NH3-N even in nitrate-supplemented lambs in this study was
sub-optimal, although it still increased microbial growth and activity as indicated by
Chapter 4: Defaunation and nitrate effects on lamb productivity
100
greater total VFA compared to unsupplemented lambs. Lambs supplemented with NO3
had an ADG of 52 g/d and grew 683 µg/cm2.d
of CWG, suggesting high efficiency of
nutrient utilisation by lambs on NO3 supplementation. Lambs without NO3 lost 25 g
LW/d and CWG grew 475 µg/cm2.d, suggesting that wool growth was utilising amino
acids as a priority over body growth.
Defaunation resulted in a significant reduction of rumen NH3-N concentration as less
digestion of engulfed feed-protein and bacteria occurs in the absence of protozoa; this
agreed with previous assessments (Jouany et al. 1988; Eugène et al. 2004a; Santra et al.
2007a; Morgavi et al. 2012). Defaunated lambs in this study had 13.8 mg NH3-N/L
rumen fluid, which was below the requirement for the maximum growth of microbes
(Satter and Slyter 1974) and thus inadequate NH3-N availability inhibited ruminal
fermentation (Leng 1990). This resulted in lower total VFA, DMD and longer MRT, but
no changes in MCP outflow, ADG or CWG by defaunated lambs. This contrasts with
previous results where defaunation increased rumen bacterial outflow and increased the
availability of protein at the duodenum (Bird and Leng 1978; Jouany 1996).
The 30% increase in DMI by nitrate-supplemented lambs was probably due to increased
NH3-N and fermentation. The negative correlation between DMI and particulate and
soluble MRT (r2= -0.52 and -0.67; P < 0.05) may reflect the positive role of NH3 in
stimulating feed breakdown in the rumen and enabling additional feed intake. The
shorter MRT in NO3 fed lambs allowed these animals to consume more feed due to a
reduced rumen fill constraint, faster passage and greater fermentation. In addition,
greater whole tract DMD would have increased CP and ME intake supporting the
Chapter 4: Defaunation and nitrate effects on lamb productivity
101
suggestion of Leng (1990) that harvesting of nutrients from low-quality forages can be
improved by ruminants if microbes in the rumen grow efficiently. This is in keeping
with the finding that lambs supplemented with NO3 in this study had higher ADG and
CWG than unsupplemented lambs. In contrast, NO3 did not increase DMI, DMD and
ADG in previous studies where protein was above ruminal requirement (van Zijderveld
et al. 2010; Li et al. 2012; de Raphélis-Soissan et al. 2014). However, because those
studies aimed to replace urea to NO3 in nitrogen-adequate diets, the authors were
unlikely to observe positive effects of NO3 on fermentation and productivity of animals
as reported here in the protein deficient diet.
4.4.3 Effects of nitrate supplementation and defaunation on methane
emissions and rumen fermentation
The higher DMP in nitrate-supplemented lambs contrasts with results from protein
adequate diets and was a consequence of higher DMI and increased ruminal
fermentation as evidenced by higher total VFA concentration and DMD, leading to
greater ruminal H2 availability. In faunated lambs, the 24.8% reduction in MY by NO3
supplementation agrees with previous studies which have shown CH4 mitigation ranges
between 23 and 35% when 1.9 to 2.6% NO3 were supplemented (Nolan et al. 2010; van
Zijderveld et al. 2010; Li et al. 2012). A review by Leng and Preston (2010) showed
that CH4 can be reduced by 16 to 50% depending on diets and the inclusion rate of NO3.
As the same amount of H2 is used to reduce 1 mol of NO3 to NH3 as 1 mol of CO2 to
CH4 (Nolan et al. 2010), the faunated lambs in this study were given 2% NO3 (14.3 g
NO3 per day during the restricted intake period), which theoretically could reduce 0.23
Chapter 4: Defaunation and nitrate effects on lamb productivity
102
mol or 4.92 g CH4/kg DMI. In this study, a reduction of 4.68 g CH4/kg DMI was
measured, which is 95% of the expected reduction, showing that most of the calcium
NO3 was reduced to NH3-N. Nitrate caused changes in rumen fermentation shifting to
increased acetate and decreased propionate as high affinity H2 of NO3 is more
favourable in NO3 reduction than in formation of propionate or CH4 (Ungerfeld and
Kohn 2006). The present study showed a tendency of lower propionate and higher
molar ratio of acetate to propionate, which was consistent with previous observations by
Nolan et al. (2010). The reduced CH4 production by NO3 supplementation may also be
a consequence of inhibiting methanogens (van Zijderveld et al. 2010) as the reduction
of NO3 to NO2 and then to NH3 resulted in a metabolic H2 sink, which decreased H2
availability for methanogens (van Zijderveld et al. 2011).
The reduced DMP after defaunation is consistent with Hegarty (1999) and can be
explained by fermentation shifting to a greater proportion of propionate and decreasing
the proportion of butyrate. However, results from this study showed DMP was reduced
with decreased total VFA and propionate proportion, but an increased acetate
proportion. A higher proportion of acetate and lower proportion of propionate in
defaunated animals fed low quality diets was also reported by Bird (1982). The lower
DMP in defaunated lambs could be due to restricted growth of microbes in the rumen,
evidenced by the lower fermentation, NH3 concentration and DMD of defaunated
lambs. Alternatively, by removing the endo-symbiotic and ecto-symbiotic methanogens
associated with protozoa, H2 may have accumulated, stimulating reductive acetogenesis
(Ungerfeld 2013). Fonty et al. (2007) also reported that reductive acetogens established
in the rumen lacking methanogens and can replace methanogens as a sink for H2 in the
Chapter 4: Defaunation and nitrate effects on lamb productivity
103
rumen, thus reductive acetogens can be potentially important to reduce enteric CH4
emissions (Joblin 1999). Because reductive acetogenesis involves the reduction of CO2
by H2 to acetate (Ungerfeld 2013), this might explain the low CH4 emissions, but high
acetate concentration in defaunated lambs. However, as H2 concentration and
methanogen population were not measured in this study, it is not possible to confirm
that hypothesis.
4.4.4 Interaction of defaunation and nitrate supplementation
In the present study, significant interactions of protozoa and NO3 occurred for MetHb
and MY. Concerning the role of protozoa on NO3/NO2 reduction and MetHb, the
present study confirmed the in vitro study by Lin et al. (2011) and showed that blood
MetHb became greater in defaunated lambs after 85 days of feeding NO3, which was
also in agreement with a study by Nakamura and Yoshida (1991) who reported that NO3
disappearance rates and blood MetHb were rapidly decreased in faunated animals
compared to defaunated animals. In feeding NO3 to defaunated and faunated lambs in
this study it was hypothesised that dietary NO3 in combination with defaunation
treatment would additively decrease methanogens and DMP. Methanogens use H2 as a
substrate for their metabolism and the use of H2 by bacteria to reduce NO3 to NO2 and
then NH3 can cause lower numbers of methanogens (van Zijderveld et al. 2010; van
Zijderveld et al. 2011). The combined treatment of NO3 and defaunation could be
expected to cause greater effects on reducing methanogen population. A significant
interaction between protozoa and NO3 supplementation on MY occurred suggesting that
Chapter 4: Defaunation and nitrate effects on lamb productivity
104
NO3 supplemented to defaunated lambs would be positively additive in lowering MY
compared to faunated lambs with or without NO3 supplementation.
4.5 Conclusion
Nitrate is an effective NPN source for rumen microbes, especially in a protein deficient
diet. From the point view of greenhouse gas mitigation, NO3 is an effective strategy to
reduced enteric CH4 emissions, provided it is supplemented at appropriate levels.
Defaunation reduced fermentation, DMP and digestion with no changes in MCP
outflow or ADG. Moreover, fermentation and digestion of defaunated lambs were
increased by NO3 supplementation and the combined treatments of defaunation and
NO3 were additive in reducing CH4 yield. This needs further investigation as combining
two CH4 mitigation strategies may be an effective approach in delivering significant
methane mitigation by grazing livestock.
Chapter 4: Defaunation and nitrate effects on lamb productivity
105
Higher Degree Research Thesis by Publication
University of New England
Statement of Originality
(To appear at the end of each thesis chapter submitted as an article/paper)
We, the PhD candidate and the candidate’s Principal Supervisor, certify that the
following text, figures and diagrams are the candidate’s original work.
Type of work
Paper numbers
Journal article 83-104
Name of Candidate: Son Hung Nguyen
Name/title of Principal Supervisor: Prof. Roger Stephen Hegarty
__________________ 14 June 2016
Candidate Date
_________________ 14 June 2016
Principal Supervisor Date
Chapter 4: Defaunation and nitrate effects on lamb productivity
106
Higher Degree Research Thesis by Publication
University of New England
STATEMENT OF AUTHORS’ CONTRIBUTION
(To appear at the end of each thesis chapter submitted as an article/paper)
We, the PhD candidate and the candidate’s Principal Supervisor, certify that all co-authors
have consented to their work being included in the thesis and they have accepted the
candidate’s contribution as indicated in the Statement of Originality.
Author’s Name (please print clearly) % of contribution
Candidate Son Hung Nguyen
85%
Other Authors Mark Barnett
5%
Roger Stephen Hegarty
10%
Name of Candidate: Son Hung Nguyen
Name/title of Principal Supervisor: Prof. Roger Stephen Hegarty
__________________ 14 June 2016
Candidate Date
_________________ 14 June 2016
Principal Supervisor Date
Chapter 5: Defaunation and nitrate effects on in vitro methane production
107
Chapter 5
Effects of rumen protozoa of Brahman heifers
and nitrate on fermentation and in vitro methane
production
S. H. Nguyena, b
, L. Lia and R. S. Hegarty
a
a School of Environmental and Rural Science, University of New England, Armidale,
NSW 2351, Australia
b National Institute of Animal Sciences, Thuy Phuong, Tu Liem, Hanoi, Vietnam
Asian-Australasian Journal of Animal Science, 2016, 29. 807-813
Chapter 5: Defaunation and nitrate effects on in vitro methane production
108
Abstract
Two experiments were conducted assessing the effects of presence or absence of rumen
protozoa and dietary nitrate addition on rumen fermentation characteristics and methane
(CH4) production in Brahman heifers. The first experiment assessed changes in rumen
fermentation pattern and in vitro CH4 production post-refaunation and the second
experiment investigated whether addition of nitrate to the incubation would give rise to
CH4 mitigation additional to that contributed by defaunation. Ten Brahman heifers were
progressively adapted to a diet containing 4.5% coconut oil distillate (COD) for 18 days
and then all heifers were defaunated using sodium 1-(2-sulfonatooxyethoxy) dodecane
(Empicol). After 15 days, the heifers were given a second dose of Empicol. Fifteen days
after the second dosing, all heifers were allocated to defaunated or refaunated groups by
stratified randomisation based on liveweight, and the experiment commenced (day 0).
On day 0, an oral dose of rumen fluid collected from unrelated faunated cattle was used
to inoculate 5 heifers and form a refaunated group so that the effects of re-establishment
of protozoa on fermentation characteristics could be investigated. Samples of rumen
fluid collected from each animal using oesophageal intubation before feeding on days 0,
7, 14 and 21 were incubated for in vitro CH4 production. On day 35, 2% of nitrate (as
NaNO3) was included in in vitro incubations to test for additivity of nitrate and of
protozoa effects on fermentation and CH4 production. It was concluded that increasing
protozoal numbers were associated with increased CH4 production in refaunated heifers
7, 14 and 21 days after refaunation. Methane production rate was significantly higher
from refaunated heifers than from defaunated heifers 35 days after refaunation.
Concentration and proportions of major volatile fatty acids, however, were not affected
Chapter 5: Defaunation and nitrate effects on in vitro methane production
109
by protozoal treatments. There is scope for further reducing CH4 output through
combining defaunation and dietary nitrate as the addition of nitrate in the defaunated
heifers resulted in 86% reduction in CH4 production in vitro.
Key words: Defaunation, refaunation, nitrate, methane production.
5.1 Introduction
Reviews of the effects of enteric protozoa on digestion and productivity by ruminants
have concluded removal of rumen ciliate protozoa reduces enteric methane (CH4)
emissions by 11% (Newbold et al. 2015) and increases an average daily gain by 11%
(Eugène et al. 2004a). Finlay et al. (1994) concluded that methanogens existing as
endo- and ecto-symbionts with ciliate protozoa contributed 37% of rumen CH4
production and Stumm et al. (1982) identified that 10 to 20% of rumen methanogens
were attached on the outside of protozoa. Centrifuging rumen fluid to remove protozoa
reduced the methanogen population by 78% (Newbold et al. 1995).
Methane production is positively related to the size of the rumen protozoal population
(Morgavi et al. 2010) and the absence of protozoa reduces CH4 production and
significantly modifies fermentation characteristics in-vitro (Qin et al. 2012). However,
Ranilla et al. (2007) reported that there was no correlation between methanogenesis and
protozoal biomass per unit of feed degraded in-vitro. Further, Bird et al. (2008) showed
that defaunation did not change enteric CH4 production 10 to 25 weeks post-treatment.
Hegarty et al. (2008) also reported that rumen protozoa did not affect CH4 production
by lambs raised without protozoa from birth, or defaunated at weaning. Therefore, the
relationship between rumen protozoa and enteric CH4 production is unclear.
Chapter 5: Defaunation and nitrate effects on in vitro methane production
110
In contrast, dietary nitrate reduces CH4 production reliably and predictably (van
Zijderveld et al. 2010; van Zijderveld et al. 2011). Nitrate reduces total gas production
when rumen fluid is incubated in vitro, and it changes the volatile fatty acid (VFA)
profile by increasing acetate and reducing propionate and butyrate molar proportions
while total volatile fatty acid concentration is unaffected (Lin et al. 2011). The
objectives of these studies were to describe the fermentation characteristics and CH4
production changes occurring in the period after refaunation of previously protozoa-free
heifers, and assess whether nitrate could further reduce CH4 production from defaunated
animals.
5.2 Materials and methods
5.2.1 Animals and feeding
All protocols for treatment and care of the cattle were approved by the University of
New England Animal Ethics Committee (AEC 13-054). Ten Brahman heifers (8 months
of age) with an average liveweight of 274 ± 32.8 kg were used. Cattle were adapted to a
pre-experimental diet of oaten (70%) and lucerne (30%) chaff with initial inclusion of
1% of coconut oil distillate (COD) which was raised to a final level of 4.5% over 8
days. Cattle were then changed to an experimental diet for 10 days to eliminate rumen
protozoa comprising oaten chaff (70%), lucerne chaff (21%), COD (4.5%) and molasses
(4.5%), resulting in 88.1% DM in the mixed ration and 7.9 % crude protein and 5%
crude fat in the dry matter. This combined 18 day period of COD dietary treatment
reduced the protozoal population from 3.91×105 cells/mL to 0.58×10
5 cells/mL of
rumen fluid and all cattle were then treated with a chemical to defaunate. After the
Chapter 5: Defaunation and nitrate effects on in vitro methane production
111
defaunation treatment, all cattle were given a diet of oaten (70%) and lucerne chaff
(30%) which included 10.5% crude protein; 1.3 crude fat; 88.8% dry matter for the
remainder of the study. All cattle had ad libitum access to the ration and water.
5.2.2 Defaunation of cattle
After 18 days feeding COD, all feed was withdrawn for a day and cattle were orally
dosed with sodium 1-(2-sulfonatooxyethoxy) dodecane (Empicol ESB/70AV, Albright
and Wilson Australia Ltd, Melbourne) administered at 45g/d in a 10% v/v solution to
remove protozoa. Cattle were dosed on three consecutive days and feed was withheld
during this treatment protocol, which was based on that of Bird and Light (2013).
Animals required 15 days to fully recover their previous voluntary intake and received
the COD diet during this period of time. The three day dosing with Empicol was then
repeated commencing 15 days after the first dosing. A further 15 days after the second
drenching program, rumen fluid samples were collected for protozoa enumeration and
the experiment commenced (d 0).
5.2.3 Refaunation of cattle
On day 0 all cattle had recovered their intake and wellbeing, and rumen fluid of the
animals was observed to be free of protozoa. Cattle were allocated to defaunated (n=5)
and refaunated groups (n=5) by stratified randomisation based on liveweight. A single
oral dose (500 mL/heifer) of a mixed rumen fluid collected from two cannulated
faunated cattle grazing pasture was used to refaunate 5 heifers. The protozoal
population in the inoculum (3.42×105
cells/mL) consisted of large holotrichs (0.13×105
Chapter 5: Defaunation and nitrate effects on in vitro methane production
112
cells/mL), small holotrichs (0.5×105
cells/mL) and small entodiniomorphs (2.79×105
cells/mL).
5.2.4 Rumen fluid sampling, ammonia, volatile fatty acid
concentrations, and protozoal enumeration
In Experiment 1, samples of rumen fluid (40 mL) were collected using oesophageal
intubation from defaunated and refaunated heifers before feeding on days 0, 7, 14 and
21. Samples from defaunated heifers were immediately checked under a microscope to
confirm that defaunated heifers were protozoa-free. Rumen pH was measured
immediately using a portable pH meter (Orion 230 Aplus, Thermo scientific, Beverly,
MA, USA). A 15 mL subsample was placed in wide-neck McCartney bottle acidified
with 0.25 mL of 18 M sulphuric acid and stored at -200C for VFA and ammonia (NH3)
analyses. A 4 mL subsample was placed in wide-neck McCartney bottle containing 16
mL of formaldehyde-saline (4% formalin v/v) for protozoa enumeration. Protozoa were
counted using a Fuchs–Rosenthal optic counting chamber (0.0625 mm2 and 0.2 mm of
depth) using a staining technique adapted from the procedure described by Dehority
(1984). The protozoa were differentiated into large (>100 µm) and small (<100 µm)
holotrichs and entodiniomorphs. Another 20 mL of subsample from defaunated and
refaunated heifers was used to conduct in vitro incubations for CH4 measurement.
In Experiment 2, samples of rumen fluid (~20 mL) were collected on day 35 using
oesophageal intubation from defaunated and refaunated heifers before feeding with each
sample being processed individually and its incubation started immediately after
collection.
Chapter 5: Defaunation and nitrate effects on in vitro methane production
113
Concentration of VFA were determined (Nolan et al. 2010) using a Varian CP 3800 Gas
Chromatograph (Varian Inc. Palo Alto, California USA) and NH3 concentration was
analysed using a modified Berthelot reaction using a continuous flow analyser (San++
,
Skalar, Breda, The Netherlands).
5.2.5 In vitro incubations and measurements
In vitro incubations (23h) were conducted using rumen fluid collected from defaunated
and refaunated heifers on days 0, 7, 14 and 21 after refaunation, to assess changes in
CH4 production in defaunated heifers and refaunated heifers while rumen protozoa were
re-establishing in refaunated heifers (Experiment 1). Samples were then taken on day 35
and incubated in vitro with the addition of 2% nitrate (NO3 as NaNO3) to test for
additivity of NO3 and defaunation effects on fermentation and CH4 production
(Experiment 2). The NaNO3 was dissolved in purified water and added in buffer
solution. The composition of incubation buffer was adapted and modified after (Soliva
and Hess 2007). For all in vitro incubations, 20 mL of rumen fluid from each animal
was injected into a Schott bottle (100 mL) which contained 40 mL of buffer solution
under a constant flow of anaerobic CO2 in a water bath maintained at 390C. Mixed
rumen fluid and buffer solution (10mL) was transferred into three 50 mL syringes (Luer
lock: Terumo Corporation, Japan) which contained 200 ± 20 mg of ground substrate
(70% oaten and 30% lucerne chaff). The syringes were sealed by a 3-way tap, pre-
warmed to 390
C and then incubated in a shaking water bath at 390
C. After the
incubations, gas volume was measured, liquid was drained from the syringes and placed
in wide-neck McCartney bottle acidified with 0.25 mL of 18 M sulphuric acid and
Chapter 5: Defaunation and nitrate effects on in vitro methane production
114
stored at -200C for VFA and NH3 analyses. The gas in the syringes were analysed for
CH4 concentration using a gas chromatograph (SMARTGAS, Varian CP 4900).
5.2.6 Statistical analyses
Data were statistically analysed using SAS 9.0 (SAS Institute, Cary, NC). Data from
Experiment 1 were subject to repeated-measures analysis of variance with protozoa,
time and protozoa × time interaction as fixed factors. Data from Experiment 2 were
subject to analysis of variance in PROC GLM, factors being protozoa, NO3 and
protozoa × NO3 interaction. Means were analysed using the least squares means
(LSMEANS) procedure. A probability of < 5% was considered to be statistically
significant.
5.3 Results
5.3.1 Protozoal population in refaunated heifers
Protozoa were not observed in any rumen fluid samples collected from defaunated
heifers during this study. In refaunated heifers, however, the protozoal population
reached 3.70×105 cells/mL by day 7 and almost doubled by day 21 (7.01×10
5 cells/mL).
Small entodiniomorphs were predominant in the total population, accounting for 94, 82
and 86 % of the total counts at days 7, 14 and 21, respectively (Figure 5.1). Methane
production from refaunated cattle was positively correlated with protozoal numbers
although CH4 production tended to stabilise after day 14 (Figure 5.2).
Chapter 5: Defaunation and nitrate effects on in vitro methane production
116
5.3.2 Fermentation pattern and methane production in Experiment 1
The rumen fluid pH was higher in refaunated heifers, but increased from day 0 to day
21 in both defaunated and refaunated heifers, showing effects of protozoal treatments
and time (Table 5.1). Ammonia concentrations increased steadily up to day 7 in both
defaunated and refaunated heifers, but refaunated heifers had higher NH3 concentrations
than did defaunated heifers (P < 0.05). Neither VFA concentration, nor molar
proportions of acetate, propionate and butyrate in total VFA, or acetate to propionate
ratio were affected by protozoal treatment, but all except butyrate proportion increased
over time.
There was an increase in total gas production in vitro by rumen fluid collected from
both defaunated and refaunated heifers from day 0 to day 14 with no significant further
increase to day 21. There was a tendency towards a lower CH4 production from rumen
fluid of defaunated heifers than from refaunated heifers over time (P = 0.07). No
significant interaction between protozoal treatment and time were observed (P > 0.05).
Chapter 5: Defaunation and nitrate effects on in vitro methane production
117
Table 5.1 The pH, ammonia concentration and concentration and molar proportions of major volatile fatty acids (VFA) in rumen
fluid, and changes in gas and methane production in vitro after refaunation.
Itema
Treatment
s.e.m
P-values
-P (n = 6) +P (n = 6)
P Day P × Day
effect Day 0 Day 7 Day 14 Day 21 Day 0 Day 7 Day 14 Day 21
pH 6.41
6.46
6.87
6.83
6.62
6.69
6.86
6.91
0.10 0.02 <0.001 0.34
NH3-N (mg/L) 32.68
30.76
59.04 62.92 36.88 69.52 86.24 117.00 9.56 <0.01 <0.001 0.08
Total VFA(mM/L) 64.43 59.67 50.92 57.95 59.46 63.43 63.03 58.16 8.05 0.63 0.18 0.39
Acetate (molar %) 71.06 74.55 75.32 79.01 73.67 73.49 73.39 76.74 1.76 0.59 0.04 0.51
Propionate (molar %) 19.15 16.61 15.05 14.46 17.75 15.66 14.52 12.30 1.40 0.12 0.02 0.95
Butyrate (molar %) 8.38 7.05 6.54 6.39 6.77 8.03 8.44 7.39 0.65 0.37 0.60 0.03
Acetate /propionate 4.07 4.65 5.08 5.57 4.58 4.77 5.07 6.29 0.51 0.26 0.44 0.90
Total gasb (mL/g DM) 102.33
128.67
144.07
157.00 103.67
135.67
152.00
149.33 4.71 0.55 <0.001 0.34
CH4 (mL/g DM) 6.44
13.60
16.86
20.66
6.99
16.76
21.68
21.47
1.29 0.07 <0.001 0.19 -P (defaunated), +P (refaunated); Standard error of the mean (s.e.m); a pH, ammonia and VFA analyses on samples collected from animals on days 0, 7, 14 and 21; b Gas and methane
production data collected from in-vitro incubations.
Chapter 5: Defaunation and nitrate effects on in vitro methane production
118
5.3.3 Fermentation pattern and methane production in Experiment 2
The pH after incubation was increased by the presence of protozoa and by NO3 (P <
0.05; Table 5.2). Ammonia concentration was also increased by the presence of
protozoa and by NO3 (P < 0.05). The presence of protozoa had little effect on VFA,
with total VFA concentration tending to be lower in rumen fluid from defaunated than
refaunated heifers, but VFA proportions were unaffected. Total VFA concentration was
significantly reduced by NO3 and a significant reduction in butyrate percentage also
occurred.
Table 5.2 The pH, ammonia concentration, volatile fatty acid concentration and molar
proportions and methane production as influenced by the presence or absence of
protozoa or nitrate addition in incubations of rumen fluid in vitro.
Parameter
Treatment
s.e.m
P-Values
-P (n =6)
+P (n =6)
P NO3 P × NO3
-NO3
+NO3 -NO3 +NO3
pH 6.19
6.49
6.02
6.32
0.05 <.01 <.01 0.98
NH3-N (mg/L) 101.19
185.71
167.23
211.70
11.60 0.01 0.01 0.18
Total VFA (mM/L) 102.96
83.15
137.57
98.01
12.14 0.08 0.04 0.14
Acetate (molar %) 69.33 70.30 67.75 68.74 1.95 0.45 0.63 0.10
Propionate (molar %) 20.34 22.48
19.57 21.54 1.14 0.47 0.11 0.94
Butyrate (molar %) 9.57
6.78
10.96
8.49
1.10 0.20 0.04 0.89
Acetate/propionate 3.42 3.15 3.47 3.26 0.26 0.76 0.39 0.92
Total gas (mL/g DM) 155.00
101.67
149.44
117.78
4.14 0.21 <.01 0.01
CH4 (mL/g DM) 18.59
3.00
22.11
12.73
0.63 <.01 <.01 <.01
Standard error of the mean (s.e.m); -P (defaunated), +P (refaunated).
Chapter 5: Defaunation and nitrate effects on in vitro fermentation
119
Methane production was reduced by both defaunation and by NO3, and there was a
significant interaction between defaunation and NO3 such that mitigation resulting from
NO3 and defaunation was greater than the mitigation resulting from either alone (P <
0.05). Methane production from defaunated heifers was lower than from refaunated
heifers (18.59 v 22.11 mL/g DM). While NO3 reduced CH4 production in refaunated
heifers (12.73 v 22.11 mL/g DM), the combined effects of defaunation and dietary NO3
on CH4 mitigation (19.11 mL) was greater than the sum of effects of defaunation (3.52
mL) and NO3 (9.38 mL) alone, implying the combined treatments were synergistic in
their mitigation potential. Total gas production was not affected by protozoal treatments
(P > 0.05), but was reduced in incubations containing NO3 (P < 0.05).
5.4 Discussion
The objectives of this study were to describe the changes in CH4 production and rumen
fermentation characteristics associated with the reintroduction of protozoa into
previously protozoa-free cattle and also assess whether CH4 mitigation arising from
NO3 would be additive to that caused by the absence of protozoa. The protozoal
population in previously defaunated heifers was established by day 7 and reached
7.01×105 cells/mL by day 21 comparable with that found by Morgavi et al. (2008) in
sheep. These authors demonstrated that total protozoal population reached their peak at
12×105 cells/mL at 25 to 30 days after inoculation and then stabilised at 7.6×10
5
cells/mL from day 60. During the refaunation period there was a substantial increase in
CH4 production rate; this result was in accordance with the positive correlation between
protozoa and CH4 production found by Morgavi et al. (2010) and the fact methanogens
that are normally attached to protozoa (Newbold et al. 1995) are responsible for 37% of
Chapter 5: Defaunation and nitrate effects on in vitro fermentation
120
rumen CH4 emissions (Finlay et al. 1994). The present study also showed that rumen
fluid from defaunated heifers tended to have a lower CH4 production in vitro than
samples from refaunated heifers 7, 14 and 21 days after refaunation. This effect may
not be exclusively a direct consequence of protozoa but also an indirect consequence of
differences in bacterial and fungal populations in the presence of protozoa (Eugène et
al. 2004a) and in some cases, an increase in activity of H2 producers (Morgavi et al.
2012). Such compensatory changes in microbial populations leading to an unchanged
VFA pattern may explain why the absence of protozoa has caused no significant
changes in CH4 emissions in defaunated animals as observed from some previous
studies (Bird et al. 2008; Hegarty et al. 2008; Morgavi et al. 2012).
Effects of protozoa on rumen NH3 concentrations are generally more consistent than
effects on VFA concentration with the concentration of NH3 lower in defaunated
ruminants compared to faunated or refaunated ones in this and previous studies (Jouany
et al. 1988; Eugène et al. 2004a; Santra et al. 2007a; Morgavi et al. 2012). Defaunation
has sometimes increased total VFA concentration in defaunated sheep (Santra et al.
2007a) and weaner lambs (Santra and Karim 2002), but Hegarty et al. (2008) found
total VFA was lower and the proportion of propionate was reduced in the protozoa-free
lambs born from defaunated ewes. These authors suggested that effects of defaunation
on reducing CH4 production may be dependent upon fermentation shifting to a more
propionate rich pattern in defaunated animals. This is consistent with defaunation
normally increasing the proportion of propionate and decreasing the proportion of
butyrate while concomitantly reducing CH4 output (Eugène et al. 2004a; Morgavi et al.
2012). No differences between defaunated and refaunated heifers in concentration and
Chapter 5: Defaunation and nitrate effects on in vitro fermentation
121
proportions of VFA were observed in these studies but the absence of protozoa still
reduced CH4 production, indicating that protozoal effects on methanogenesis are not
just a consequence of increased partitioning of H2 into propionate synthesis.
Importantly, the successive in vitro studies show that despite defaunation being
completed 15 days before day 0; the rumen of defaunated heifers was not metabolically
stable, with pH, total VFA, NH3 and acetate percentage changing out to day 21 in
Experiment 1. Little is known about rumen ecological stabilisation after defaunation, it
was presumable in these studies that rumen ecology was stable within 50 days after
defaunation and therefore was stable when the combined effects of NO3 and defaunation
were assessed in the Experiment 2.
Dietary NO3 has been shown to offer a reliable and predictable strategy to mitigate CH4
production from ruminants in both in vitro and in vivo studies. A review by Leng and
Preston (2010) concluded that the use of NO3 as a H2 sink could reduce CH4 production
from 16-50%, depending on diets and the inclusion rate of NO3. This is because
approximately 2 mol of H2 will be needed to convert NO3 to nitrite and 6 mol of H2
will be removed in order to reduce nitrite to NH3 (Allison and Reddy 1984). The result
from the Experiment 2 shows that CH4 production was significantly lowered by
addition of NO3 in refaunated heifers 35 days after refaunation, confirming the potential
for role of dietary NO3 as a strategy to mitigate CH4 emissions (Guo et al. 2009; Nolan
et al. 2010; van Zijderveld et al. 2010; van Zijderveld et al. 2011). In addition, NO3
reduced total gas production, total in vitro VFA concentrations and the proportion of
butyrate in line with findings of Lin et al. (2011). The present study also indicated that
Chapter 5: Defaunation and nitrate effects on in vitro fermentation
122
the combined effects of protozoal treatment and dietary NO3 led to more than additive
reduction in CH4 production (19.11 mL) compared with the sum of the protozoal effect
(3.52 mL) and the dietary NO3 effect (9.38 mL).
5.5 Conclusion
Methane production was positively correlated with protozoal numbers in rumen fluid in
the period following refaunation of defaunated heifers with protozoa (Experiment 1).
The absence of protozoa reduced CH4 production by 16% compared with refaunated
heifers, and the combined effects of NO3 and defaunation was synergistic in CH4
mitigation (Experiment 2). Future research is needed to confirm these suggestions and
gain better understandings the changes in gut fermentation, adaptation of methanogen
and increased activity of some rumen microbes after defaunation and refaunation. In-
vivo experiments need to be undertaken to gain a better understanding of the combined
effects of defaunation and dietary NO3 on CH4 production in cattle.
Chapter 5: Defaunation and nitrate effects on in vitro fermentation
123
Higher Degree Research Thesis by Publication
University of New England
Statement of Originality
(To appear at the end of each thesis chapter submitted as an article/paper)
We, the PhD candidate and the candidate’s Principal Supervisor, certify that the
following text, figures and diagrams are the candidate’s original work.
Type of work
Paper numbers
Journal article 107-122
Name of Candidate: Son Hung Nguyen
Name/title of Principal Supervisor: Prof. Roger Stephen Hegarty
__________________ 14 June 2016
Candidate Date
_________________ 14 June 2016
Principal Supervisor Date
Chapter 5: Defaunation and nitrate effects on in vitro fermentation
124
Higher Degree Research Thesis by Publication
University of New England
STATEMENT OF AUTHORS’ CONTRIBUTION
(To appear at the end of each thesis chapter submitted as an article/paper)
We, the PhD candidate and the candidate’s Principal Supervisor, certify that all co-authors
have consented to their work being included in the thesis and they have accepted the
candidate’s contribution as indicated in the Statement of Originality.
Author’s Name (please print clearly) % of contribution
Candidate Son Hung Nguyen
85%
Other Authors Lily Li
5%
Roger Stephen Hegarty
10%
Name of Candidate: Son Hung Nguyen
Name/title of Principal Supervisor: Prof. Roger Stephen Hegarty
__________________ 14 June 2016
Candidate Date
_________________ 14 June 2016
Principal Supervisor Date
Chapter 6: Defaunation and oil effects on rumen fermentation
125
Chapter 6
Effects of defaunation and dietary coconut oil
distillate on fermentation, digesta kinetics and
methane production of Brahman heifers
S. H. Nguyena, b
and R. S. Hegartya
a School of Environmental and Rural Science, University of New England, Armidale,
NSW 2351, Australia
b National Institute of Animal Sciences, Thuy Phuong, Tu Liem, Hanoi, Vietnam
Journal of Animal Physiology and Animal Nutrition (accepted April, 2016)
Chapter 6: Defaunation and oil effects on rumen fermentation
126
Abstract
A 2×2 factorial experiment was conducted to assess the effects of presence or absence
of rumen protozoa and of dietary coconut oil distillate (COD) supplementation on
rumen fermentation characteristics, digesta kinetics and methane (CH4) production in
Brahman heifers. Twelve Brahman heifers were selected to defaunate, with 6 being
subsequently refaunated. After defaunation and refaunation, heifers were randomly
allocated to COD supplement or no supplement treatments while fed an oaten chaff
basal diet. Daily methane production (DMP; 94.17 v 104.72 g/d) and methane yield
(MY; 19.45 v 21.64 g/dry matter intake) were reduced in defaunated heifers compared
to refaunated heifers when measured 5 weeks after refaunation treatment (P < 0.01).
Supplement of COD similarly reduced DMP and MY (89.36 v 109.53 g/d and 18.46 v
22.63 g/kg dry matter intake respectively; P < 0.01) and there were no significant
interactions of defaunation and COD effects on rumen fermentation or CH4 emissions.
Concentration of volatile fatty acids and molar proportions of acetate, propionate and
butyrate were not affected by defaunation or by COD. Microbial crude protein outflow
was increased by defaunation (P < 0.01) in the absence of COD but was unaffected by
defaunation in COD supplemented heifers. There was a tendency towards a greater
average daily gain in defaunated heifers (P = 0.09), but COD did not increase average
daily gain (P > 0.05). The results confirmed that defaunation and COD independently
reduced enteric DMP even though the reduced emissions were achieved without altering
rumen fermentation volatile fatty acid levels or gut digesta kinetics.
Keywords: Protozoa, methanogensis, fatty acids, cattle.
Chapter 6: Defaunation and oil effects on rumen fermentation
127
6.1 Introduction
Of the 87.4 Mt CO2-e total greenhouse gas emissions released annually from Australia’s
agricultural sector, 64% are from enteric fermentation of livestock (Department of the
Environment 2014). Enteric methane (CH4) is not only Australia’s largest agricultural
emission source but also represents a loss of 5 to 7% of gross energy intake, equivalent
to a CH4 yield of 16 to 26 g CH4/kg of dry matter consumed (Hristov et al. 2013).
Reviews of the effects of enteric protozoa on digestion and productivity by ruminants
have concluded that removal of rumen ciliate protozoa (defaunation) reduces enteric
CH4 emissions by 11% (Newbold et al. 2015) and increases average daily gain by 11%
(Eugène et al. 2004a). Despite these average effects, there are many studies where
animals with a stable protozoa-free rumen do not exhibit reduced CH4 emissions.
Defaunation did not change enteric CH4 production 10 to 25 weeks post-treatment (Bird
et al. 2008) and did not affect CH4 production by lambs raised without protozoa from
birth or from weaning (Hegarty et al. 2008). Although, defaunation reduces the number
of methanogens in rumen fluid, it does not always reduce CH4 production (Kumar et al.
2013). Therefore, evidence that CH4 emissions are reduced in ruminants that have a
stable long-term defaunated rumen remains unclear.
The use of medium-chain fatty acids to reduce methanogen and ciliate protozoal
populations in the rumen has been shown as a potential strategy to reduce CH4
production. Coconut oil (CO) is a rich source of medium-chain unsaturated fatty acids
and feeding 3.5% and 7% CO reduced CH4 production by 28% and 73% in sheep
(Machmüller and Kreuzer 1999). Feeding 50 g CO/day significantly reduced CH4
emissions without affecting the total tract dry matter digestion or energy retention
Chapter 6: Defaunation and oil effects on rumen fermentation
128
within sheep (Machmüller et al. 2003). Jordan et al. (2006) showed that feeding 250 g
refined CO/day to beef heifers reduced CH4 output by 18%, while dry matter intake was
maintained and liveweight gain increased. However, no interaction between CO feeding
and defaunation was observed for CH4 production (Machmüller et al. 2003).
While studies of rumen ciliate protozoa effects on CH4 production and performance of
sheep have been recently published (Eugène et al. 2010; Morgavi et al. 2012; Zeitz et
al. 2012), there is little data on defaunation of cattle. Therefore, this study sought to
investigate the fermentation characteristics, digesta kinetics and CH4 emissions in
defaunated beef heifers compared to heifers with protozoa on a forage diet, and assess
whether coconut oil distillate (COD) supplementation could further reduce CH4
emissions from defaunated heifers.
6.2 Materials and methods
6.2.1 Animals and feeding
All protocols for treatment and care of the cattle were approved by the University of
New England Animal Ethics Committee (AEC 13-054). Twelve purebred Brahman
heifers (8 months of age) with an average liveweight (± s.e) of 280 ± 27 kg were
obtained and defaunated. After this defaunation program, heifers were allocated to four
experimental groups by stratified randomisation procedures based on liveweight to form
a 2×2 factorial design (protozoa status either defaunated or refaunated; COD
supplementation at 0 or 4.5%). A diet of 4.5 % COD and 4.5% molasses was prepared
by sprinkling of the liquid COD and molasses onto a mix of 70% oaten and 21%
lucerne chaff while the chaff was tossed in a rotary feed mixer (+COD; Table 6.1). As
Chapter 6: Defaunation and oil effects on rumen fermentation
129
COD was not highly palatable, molasses was added in the diet to enable consumption of
4.5% COD. The unsupplemented control diet (-COD; Table 6.1) was prepared as a
straight mix of 70% oaten and 30% lucerne hay. All heifers had ad libitum access to the
diets until 3 days prior to CH4 measurement from day 15 to day 18 (Table 6.2), at which
time feed intake was restricted to 80% of averaged ad libitum intake. Restricted feed
intake continued during a measurement period of faeces collection for analysis of
digesta kinetics from day 18 to day 22. Heifers were fed twice daily in two equal
portions at 0930 and 1500 hours. Water was always available ad libitum. All heifers
were weekly weighed to monitor liveweight and determined average daily gain (ADG).
6.2.2 Feed sampling and chemical analyses
Feed samples (~100 g) were collected before and after each mix of feed and stored at -
200C. All samples were pooled and sub-samples were taken to analyse chemical
composition (Table 6.1). Feed samples were analysed by the NSW DPI Feed Quality
Service, Wagga Wagga Agriculture Institute, NSW, Australia. Crude protein was
assessed by wet chemistry (AOAC 990.03 method), crude fat by petroleum ether
extract, metabolizable energy by the AFIA 2.2R method, dry matter digestibility,
digestible organic matter in the dry matter by wet chemistry (AFIA method 1.7R), acid
detergent fibre and neutral detergent fibre by near-infrared spectroscopy (AFIA 2014).
Chapter 6: Defaunation and oil effects on rumen fermentation
130
Table 6.1 Composition of the diets and fatty acid profile of coconut oil distillate
(g/100g dry matter).
Component (+COD)*
(-COD)†
Dry matter (g/100g as fed) 88.1 88.8
Dry matter digestibility 60 60
Digestible organic matter in dry matter 60 58
Inorganic ash 8.0 8.0
Organic matter 92 92
Neutral detergent fibre 48 52
Acid detergent fibre 28 31
Crude protein 8.2 10.3
Metabolisable energy (MJ/kg) 10.0 8.9
Crude fat 5.0 1.3
Fatty acid profile (%) of COD
C8:0
C10:0
C12:0
C14:0
C16:0
C18:0
6.15
5.00
42.08
15.69
13.41
3.50
*Oaten chaff (70%), lucerne chaff (21%), coconut oil distillate (4.5%), molasses (4.5%) fresh weight basis; †Oaten
chaff (70%), lucerne chaff (30%) fresh weight basis.
6.2.3 Defaunation of cattle
All heifers were acclimated to a diet consisting of oaten (70%) and lucerne (30%) chaff
with initial COD from 0 to 4.5% for 18 days to supress protozoa. Initial COD treatment
reduced the protozoal population from 3.91×105 cells/mL to 0.58×10
5 cells/mL of
rumen fluid. The defaunation treatment was adapted from a protocol by Bird and Light
(2013), with heifers fasted for 24 h and then orally dosed with sodium 1-(2-
sulfonatooxyethoxy) dodecane (Empicol ESB/70AV, Albright and Wilson Australia
Ltd, Melbourne) administered at 45 g/d in a 10% v/v solution to remove protozoa.
Chapter 6: Defaunation and oil effects on rumen fermentation
Heifers were dosed on three consecutive days and feed was withheld during this period.
Animals required 15 days to recover the voluntary intake observed prior to treatment.
The three day dosing with Empicol was repeated commencing 15 days after the first
dosing. Weekly rumen fluid samples were collected from all heifers for protozoa
enumeration commencing a further 15 days after the second drenching program.
Table 6.2 Experimental schedule for the defaunation, refaunation and data
measurements.
Day Activity
Defaunation
-69 Coconut oil distillate (COD) feeding period: 18 day following
8 day adaptation
-51 First three day defaunation protocol and recovery period
commencing on day 51
-36 Second three day defaunation protocol and recovery period
commencing on day 36
Refaunation
-21 Six heifers were inoculated with rumen fluid from cannulated
cattle to refaunate
-21, -14, -7,
0
Protozoa check in both groups of defaunation and refaunation,
rumen VFA and ammonia sampling
Comparative
study
0 Start of 2x2 study (COD supplementation at 0 and 4.5%;
protozoa status either defaunated or refaunated)
15 - 18 Methane production measured, rumen protozoa, VFA and NH3
sampling
18 - 22 Digesta kinetics measured, spot urine samples, rumen
protozoa, VFA and NH3 sampling
Chapter 6: Defaunation and oil effects on rumen fermentation
132
6.2.4 Refaunation of cattle
All heifers had recovered their intake and wellbeing 15 days after the second
defaunation program, and their rumen fluid was visually observed to be free of
protozoa. Six heifers were selected for refaunation by stratified randomisation based on
liveweight. A single oral dose (total of 500 mL/heifer) of a mixed rumen fluid collected
from two cannulated faunated cattle grazing pasture was used to refaunate 6 heifers. The
protozoal population in the inoculum (3.42×105
cells/mL) consisted of large holotrichs
(0.13×105
cells/mL), small holotrichs (0.5×105
cells/mL) and small entodiniomorphs
(2.79×105
cells/mL).
6.2.5 Rumen fluid sampling, ammonia, volatile fatty acid
concentrations, and protozoal enumeration
Samples of rumen fluid were collected weekly by oesophageal intubation commencing
15 days after the second defaunation program from defaunated animals to confirm the
sustained protozoa free status during the experimental period. Additional samples from
refaunated heifers were also collected to monitor the protozoal growth immediately
after inoculation. When collecting rumen fluid, rumen pH was measured immediately
upon sampling using a portable pH meter (Orion 230 Aplus, Thermo scientific, Beverly,
MA, USA). A 15 mL subsample was placed in wide-neck McCartney bottle acidified
with 0.25 mL of 18 M sulphuric acid and stored at -200C for volatile fatty acid (VFA)
and ammonia (NH3) analyses. A 4 mL subsample was placed in wide-neck McCartney
bottle containing 16 mL of formaldehyde-saline (4% formalin v/v) for protozoa
enumeration and stored at room temperature for the visual enumeration of ciliate
Chapter 6: Defaunation and oil effects on rumen fermentation
133
protozoa. Protozoa were stained prior to counting using an adaption of the procedure of
Dehority (1984). Protozoa were counted using a Fuchs–Rosenthal optic counting
chamber (0.0625 mm2 and 0.2 mm of depth). The protozoa were differentiated into
large (>100 µm) and small (<100 µm) holotrich and entodiniomorph grouping.
Concentration of VFAs was determined by gas chromatography (Nolan et al. 2010)
using a SMARTGAS Varian CP 3800 Gas Chromatograph (Varian Inc. Palo Alto,
California USA) and NH3 concentration was analysed using a modified Berthelot
reaction using a continuous flow analyser (San++
, Skalar, Breda, The Netherlands).
6.2.6 Methane production measurement
Daily methane production (DMP, g CH4/day) was measured in respiration chambers
over 2×22 h consecutive period (Hegarty et al. 2012). Heifers were placed in their
chambers by 1100 hours, with their feed and water available inside the chambers. The
chambers were opened to collect refusals and supply fresh feed at 0900 hours the
following day and only resealed at 1100 hours to commence the second 22 h of
measurement until 0900 hours the following day.
Methane recovery through each chamber was quantified immediately before and after
the experimental period. Pure CH4 was infused into each chamber at a known rate using
a mass flow meter (Smart Trak 2 Series 100, Sierra Instruments, Monterey). The
concentration of CH4 reached a plateau in the chamber after 60 mins. Knowing the
inflow rate of CH4 (CH4 infusion rate) and the chamber outflow rate (air flow rate
through the chamber) an expected plateau value for each chamber could be calculated.
A value for CH4 recovery was obtained (89-100%) from the ratio of the measured
Chapter 6: Defaunation and oil effects on rumen fermentation
134
plateau CH4 concentration and expected methane plateau value and DMP corrected
accordingly. Methane yield (MY) was calculated as DMP divided by dry matter intake
(DMI).
6.2.7 Digesta kinetics and estimation of microbial protein supply
A 5-day collection of faecal output was conducted to determine digesta kinetic and dry
matter digestibility (DMD). The mean retention time (MRT, h) of digesta was estimated
in all heifers over 5 days by reference to faecal excretion of a dosed particle-phase
marker (50 g per heifer of Cr-mordanted NDF from oaten chaff) prepared in accordance
with Udén et al. (1980), and a liquid-phase marker (60 g per heifer of Co-EDTA from
AVA Chemicals Pty Ltd. Mumbai, India in 250 mL of Milli-Q water). Dissolved Co-
EDTA was administered via intubation directly into the rumen as a single dose while
the Cr-mordanted fibre was offered to each heifer with 100 g of lucerne chaff
immediately prior to the morning feed. Faecal samples were collected at 6 h after
administration of the liquid marker, following by every 3 h for the next 48 h, every 8 h
for the next 24 h and every 12 h for the next 24 h.
Samples were analysed Cr and Co concentrations (Barnett et al. 2016) using portable X-
ray fluorescence spectroscopy (Bruker Tracer III-V pXRF, Bruker Corp, MA USA).
Analysis of digesta kinetics was undertaken using non-linear curve fitting algorithms of
WinSAAM (Aharoni et al. 1999).
During faecal marker collection, spot samples of urine were collected from all heifers.
Urine samples in 50 mL plastic bottles containing 5% H2SO4 were stored at -20 0C until
analysis. Urine allantoin and creatinine concentrations were determined (IAEA, 1997).
Chapter 6: Defaunation and oil effects on rumen fermentation
135
Microbial crude protein (MCP) outflow was estimated in spot urine samples as
calculated by the equations of Chen et al. (2004).
6.2.8 Statistical analyses
Data was statistically analysed using SAS 9.0 (SAS Institute, Cary, NC). Data from
rumen fermentation characteristics, digesta kinetics, MCP outflow, DMP and MY were
subject to analysis of variance in PROC GLM with factors being protozoa, COD and
protozoa × COD interaction. For analysis of ADG, the model used the initial liveweight
as a covariate. Homogeneity of variance and normal distribution were tested using
PROC UNIVARIATE before statistical analysis. Means were analysed using the least
squares means (LSMEANS) procedure. A probability of < 5% was considered to be
statistically significant.
6.3 Results
6.3.1 Protozoal populations
Defaunated heifers remained protozoa-free throughout the study. In refaunated heifers,
the total numbers of protozoa after refaunation reached 3.70×105/mL by day 7 and
doubled by day 21 (7.38×105/mL), which was day 0 of COD feeding period (Table 6.3).
Small entodiniomorphs were predominant in the population of rumen protozoa, ranging
from 82 to 94% of the total. The total number of protozoa and numbers of small
entodiniomorphs in refaunated heifers after 22 days of feeding COD were significantly
reduced by COD (Table 6.3) such that total numbers of protozoa and of small
entodiniomorphs were reduced by COD by 84% and 82% respectively (P < 0.05). Large
Chapter 6: Defaunation and oil effects on rumen fermentation
136
holotrichs were not affected by COD while small holotrichs were not detected after day
22 feeding COD. Large entodiniomorphs were not observed in the inoculum or in any
rumen fluid samples of refaunated heifers.
6.3.2 Rumen pH, volatile fatty acid and ammonia concentrations
Defaunation reduced rumen pH (P = 0.03) while COD supplement did not affect rumen
pH (P > 0.05; Table 6.4). Concentrations of VFA and molar proportions of acetate,
propionate, butyrate and the molar ratio of acetate to propionate were not significantly
affected by defaunation or by COD. There were no interactions between protozoal
treatments and dietary COD on concentrations of VFA or their molar proportions (P >
0.05). Defaunation and COD significantly reduced ruminal NH3-N concentration by
39% and 61% respectively (P < 0.05) and an interaction between defaunation and COD
was found (P < 0.01) such that refaunation raised ruminal NH3-N concentration in the
absence of COD, but did not affect NH3 when COD was included (Figure 6.1).
Table 6.3 Enumeration of protozoa following refaunation in heifers and fed a diet
containing either 4.5% coconut oil distillate (+COD) or nil (-COD).
Parameters
Refaunated heifers
s.e.m
P-Values
Initial (d 0)
Final (d 22)
COD Day COD
× Day -COD +COD -COD +COD
Total protozoa
(×105/mL)
7.04 7.72 6.88 1.25 1.00 0.01 0.04 0.02
Large holotrich 0.06 0.19 0.17 0.10 0.11 1.00 0.81 0.34
Small holotrich 0.47 1.17 0.61 0 0.10 <.01 0.68 <.01
Small entodiniomorph 6.52 6.36 6.10 1.17 0.92 0.02 0.02 0.03
Standard error of the mean (s.e.m).
Chapter 6: Defaunation and oil effects on rumen fermentation
138
Table 6.4 Physiological and rumen fermentation characteristics as influenced by the
presence (+P) or absence of protozoa (-P) or coconut oil distillate (±COD)
supplementation.
Parameter
Treatment
SEM
P-Values
Protozoa
COD P COD
P×
COD -P +P -COD +COD
Rumen pH 6.29 6.50 6.40 6.38 0.06 0.03 0.82 0.24
NH3-N (mg/L) 46.83 76.60 88.90 34.53 6.36 0.01 <.01 0.01
Total VFA (mM/L) 49.30 42.53 44.16 48.03 4.05 0.18 0.56 0.55
Acetate (molar %) 76.19 75.54 76.05 75.67 1.06 0.67 0.81 0.29
Propionate (molar %) 14.98 13.64
13.83 14.79 0.62 0.14 0.29 0.13
Butyrate (molar %) 6.75 7.58 6.67 7.66 0.53 0.28 0.20 0.87
Acetate / propionate
ratio 5.23 5.72 5.69 5.25 0.32 0.29 0.33 0.11
DMI (kg/day)*
5.36 5.45 6.25 4.63 0.20 0.24 <.01 0.69
DMD (%)†
46.38 45.05 49.99 41.44 3.28 0.78 0.10 0.06
DMP (g CH4/day) 94.17 104.72 109.53 89.36 3.45 <.01 <.01 0.33
MY (g CH4/kg DMI)‡
19.45 21.64 22.63 18.46 0.71 <.01 <.01 0.32
MI (g CH4/kg ADG) 125.3 245.2 223.0 147.5 60.54 0.08 0.24 0.20
ADG (kg/day)§
0.84 0.54 0.71 0.67 0.12 0.09 0.69 0.25
Final LW (kg) 313.7 301.0 308.2 306.5 7.70 0.13 0.80 0.27
FCR (g FI/g ADG)¶
7.84 14.65 13.95 8.54 2.39 0.07 0.14 0.16
MCP outflow (g/day) 85.36 66.21 80.65 70.92 6.26 <.01 0.13 0.02
Rumen soluble
MRT (h) 16.52 15.40 14.72 17.20 2.27 0.64 0.31 0.46
Hindgut soluble
MRT (h) 8.64 7.60 8.24 8.00 0.75 0.20 0.76 0.36
Standard error of the mean (s.e.m); *Dry matter intake (DMI) was calculated when cattle were on ad libitum fed; †Dry matter digestibility (DMD) was estimated based on soluble marker (Co-EDTA); ‡kg DMI was calculated when
heifers were on restricted intake; §Average daily gain (ADG) was calculated as (LW at d-21- final LW at d 22)/43
days; Daily methane production (DMP); Methane yield (MY); Methane intensity (MI).¶Feed conversion ratio (g feed
intake/g ADG).
6.3.3 Methane emissions
Daily methane production (g CH4/d) and MY (g CH4/kg DMI) were significantly
reduced by defaunation (10%) and COD (18%), respectively (P < 0.01; Table 6.4).
Combination of defaunation with COD supplement did not cause interactions in DMP
Chapter 6: Defaunation and oil effects on rumen fermentation
139
or MY, indicating that the effects were at least additive. When CH4 intensity (MI; g
CH4/kg ADG) was calculated using ADG over 43 days from defaunation to the end of
the study, defaunation tended to reduce MI (P = 0.08), but supplementation of COD did
not change MI (P = 0.24).
6.3.4 Dry matter intake, digestibility, digesta kinetics, microbial
protein outflow and liveweight change
The presence or absence of rumen protozoa did not affect DMI, but COD addition
reduced DMI (P < 0.05) as the DMI was 26% lower in heifers with COD compared to
heifers without COD. Protozoal treatment and dietary COD did not affect DMD or the
rumen or hindgut MRT of liquid digesta (P > 0.05; Table 6.4). Rumen and hindgut
particle MRT were unable to be estimated due to heifers being slow to consume the Cr-
mordanted NDF dose, making fitting of dilution curve difficult.
Defaunation increased MCP outflow by 22% (P < 0.01; Table 6.4), but MCP outflow
was not affected by COD supplementation. There was an interaction between
defaunation and COD on MCP outflow (P = 0.02, Figure 6.2), showing reduced MCP
outflow in defaunated heifers when COD was supplemented, but MCP outflow was
unaffected by COD in refaunated heifers which all had a low MCP outflow. There was a
positive correlation between MCP outflow and ADG such that higher MCP outflow was
associated with higher ADG (ADG = -0.34 + 0.014 MCP outflow, r2
= 0.67, P = 0.02).
Defaunated heifers tended to have a greater ADG (P = 0.09) and a lower feed
conversion ratio (FCR, P = 0.07) compared to refaunated heifers, while COD
Chapter 6: Defaunation and oil effects on rumen fermentation
141
In sheep, defaunation has been successfully achieved with sodium 1-(2-
sulfonatooxyethoxy) dodecane (Empicol ESB/70AV, Albright and Wilson Australia
Ltd, Melbourne) (Bird et al. 2008; Hegarty et al. 2008), but there are very few reports
of successful defaunation of cattle (Bird and Light 2013), probably because omasal
ciliate protozoa are difficult to eliminate in cattle (Towne and Nagaraja 1990). A
defaunation protocol modified from that used in sheep was developed, with preliminary
addition of COD rich in lauric acid (42%) to the diet for 18 days prior to treatment with
Empicol. This COD treatment reduced the rumen protozoal population by 85%, and
following two treatments of Empicol, rendered cattle without detectable levels of rumen
protozoa.
6.4.1 Protozoal population in refaunated heifers after inoculation and
effect of coconut oil distillate on protozoal population
The protozoal population in heifers after refaunation from a previously defaunated state
was well established by day 7 and reached 7.38×105 cells/mL by day 21, comparable
with that found by Sénaud et al. (1995) who re-inoculated defaunated rumen with
Isotricha and ciliates of mixed fauna and found that the maximum concentration of
protozoa was reached 9 to 17 days after inoculation. The maximum population then
decreased for 2-3 days before stabilising. Morgavi et al. (2008) also demonstrated that
total protozoal populations reached their peak at 12×105
cells/mL by 25 to 30 days after
inoculation and then stabilised at 7.6×105
cells/mL from day 60.
Capric acid (C10:0), lauric acid (C12:0) and myristic acid (C14:0) show strong
protozoal toxicity and are useful rumen defaunating agents (Matsumoto et al. 1991).
Chapter 6: Defaunation and oil effects on rumen fermentation
142
The present study showed that total numbers of rumen protozoa were reduced by 81%
after 22 days of feeding COD (containing 42% lauric acid) when included at 4.5% of
the diet. Matsumoto et al. (1991) observed the rumen protozoa, except Entodinium spp.
were undetectable after 3 days feeding of 30 g of hydrated CO containing 52% lauric
acid. Feeding 250g of refined CO to beef heifers reduced total protozoa by 62% (Jordan
et al. 2006) and protozoal populations in beef heifers were decreased by 63% and 80%
by 300 g/d CO after 45 and 75 days, respectively (Lovett et al. 2003). Machmüller,
(2006) observed a reduction in rumen protozoa by 88 and 97% when feeding sheep with
3.5 and 7% CO respectively. This suppressive effect of CO on rumen protozoa even
persisted 5 weeks after finishing feeding sheep with CO (Sutton et al. 1983).
6.4.2 Effects of defaunation treatment
The effects of defaunation on VFA concentration and the molar proportions of VFA are
not entirely consistent in the literature (Jouany et al. 1988; Williams and Coleman 1992;
Eugène et al. 2004a; Newbold et al. 2015), and in this study defaunation did not cause
changes in total VFA or molar proportions of acetate, propionate and butyrate.
Defaunation sometimes increased total VFA concentration in defaunated sheep (Santra
et al. 2007a) and weaner lambs (Santra and Karim 2002), but Hegarty et al. (2008)
reported that animals with protozoa had higher concentrations of total VFA compared
with defaunated animals. Molar proportions of VFA were also inconsistently affected
by defaunation as butyrate and acetate proportion were increased (Machmüller et al.
2003; Bird et al. 2008) and proportion of propionate was decreased (Machmüller et al.
2003; Hegarty et al. 2008). A higher proportion of acetate and lower proportion of
Chapter 6: Defaunation and oil effects on rumen fermentation
143
propionate in the VFA of defaunated animals was a common finding by Bird, (1982)
when animals were fed low-quality diets. These inconsistent effects of defaunation on
VFA concentration and molar proportions may reflect variable effects of defaunation on
the bacterial population in defaunated rumens. Reviews of literature by Jouany et al.
(1988) concluded that defaunation increased numbers of bacteria, which induced
changes in digestion and fermentation due to bacterial species distribution and bacterial
composition had been changed after defaunation (Ozutsumi et al. 2005).
In the present study, the reduced DMP of defaunated heifers was not associated with
increased molar proportions of propionate, which contrasted to a study by Eugène et al.
(2004a). Morgavi et al. (2012), however, reported a negative correlation between H2
concentration and CH4 production, such that less production of CH4 resulted in a high
concentration of dissolved H2, but propionate production did not increase.
Accumulation of H2 in association with lower CH4 production may reflect reduced
capacity to utilise H2 by microbes in the defaunated rumen and it is possible that
reductive acetogenic bacteria in the rumen (Joblin 1999) could potentially convert
accumulated H2 and CO2 into acetate when H2 partial pressure is raised (Fonty et al.
2007; Ungerfeld 2013). As H2 concentration and methanogen populations were not
measured in this study, it is not possible to confirm that inhibition of methanogenesis
caused accumulation of H2 or induced reductive acetogenesis.
Methanogens, which exist as endo- and ecto-symbionts with ciliate protozoa (Finlay et
al. 1994; Tokura et al. 1997), had been estimated to accout for 37% of CH4 production
(Finlay et al. 1994) and the proportions of methanogens in the total bacterial population
Chapter 6: Defaunation and oil effects on rumen fermentation
144
were lower in association with a 26% lower CH4 emissions from protozoa-free lambs
compared to faunated lambs (McAllister and Newbold 2008). In addition, the archaeal
community of methanogens in liquid and solid rumen contents were similar in faunated
wethers, but a lower proportion of methanogens in the liquid phase was associated with
defaunation (Morgavi et al. 2012). However, Mosoni et al. (2011) observed a 20%
reduction in CH4 emissions in long-term (2 year) and short-term (10 week) defaunation,
but methanogens per gram of dry matter of rumen content increased while the diversity
of dominant methanogenic community was not changed. Therefore, it may not be
reasonable to attribute the reduced CH4 production from defaunation to a loss of
methanogens (Morgavi et al. 2012). It is hypothesised that loss of ciliate-associated
methanogens in the defaunated rumen reduced CH4 production but may have induced an
increase in other populations of micro-organism, with a lower prevalence of H2
producers.
Removing protozoa from the rumen may allow a proliferation of rumen bacteria,
leading to increased uptake of NH3 by bacteria for protein synthesis and less protein
being degraded by protozoa (Williams and Coleman 1992). Decreases in NH3
concentration in defaunated animals compared to faunated or refaunated animals were
observed in this study and confirm previous assessments (Jouany et al. 1988; Eugène et
al. 2004a; Santra et al. 2007a; Morgavi et al. 2012; Newbold et al. 2015). Less ruminal
catabolism of engulfed feed-protein and bacteria occurs in the absence of protozoa,
leading to an increase in the supply of protein to the duodenum (Bird and Leng 1978;
Jouany 1996). The present study showed 22% increased MCP outflow in defaunated
heifers, which was consistent with previous studies. The increased MCP outflow was
Chapter 6: Defaunation and oil effects on rumen fermentation
145
associated with a 9-35% increased ADG in defaunated animals given high or low-
quality forage diets (Bird 1989). There was a tendency for defaunated heifers to have a
greater ADG, and this could only be ascribed to the increased MCP supply and/or
reduced loss of digested energy in CH4. Heifers in this experiment received oaten chaff
based diet providing 10 g of CP per MJ of ME, which theoretically met the microbial
protein yield required for growing cattle (CSIRO 2007). However, defaunated heifers
had a decreased NH3 concentration to below 50 mgN/L, which may have been
insufficient to maximise microbial synthesis in the rumen (Satter and Slyter 1974). This
could explain why defaunation did not more strongly increase ADG of animals given
forage based diets in this study, although defaunation increased MCP outflow.
6.4.3 Effects of coconut oil distillate supplementation
The 20% and 24% reduction in DMP and MY when refaunated heifers received 4.5%
COD (200 g/day) in this study was consistent with previous assessments (Lovett et al.
2003; Machmüller et al. 2003; Jordan et al. 2006; Machmüller 2006; Patra 2014).
Jordan et al. (2006) reported a decrease in 18% CH4 production by beef heifers that
received 250 g/day of refined CO. The reduced CH4 emissions by COD from this study
were not associated with changes in total VFA or molar proportions of acetate or
propionate. However, the relationship between feeding ruminants fat and the
proportions of VFA associated with CH4 production is not consistent with published
data. Dietary fat decreased CH4 production linearly by 4.3% per percentage of fat
inclusion and tended to shift the proportions of VFA to greater propionate and less
butyrate production in the study of Patra, (2014). In contrast, results from this study
Chapter 6: Defaunation and oil effects on rumen fermentation
146
agreed with Hristov et al. (2009) who reported no differences in total VFA when
feeding lactating cows with lauric acid and CO. Methane production was reduced by
60% by CO, but the proportion of propionate was unchanged. In the same study by
Hristov et al. (2009), lauric acid increased the proportion of propionate and decreased
the proportion of butyrate, but CH4 production was not reduced. In contrast, an in vitro
study by Machmüller et al. (2002) showed that lauric acid reduced CH4 production
(mmol per gram organic matter degraded) by 76%, but did not affect total VFA or
molar proportions of VFA. The reduced CH4 production by MCFA in this and previous
studies was most likely a result of reduced rumen protozoa and methanogens (Dohme
et al. 2000; Liu et al. 2011) and also reduced butyrate producing bacterial populations
such as Butyrivibrio fibrisolvens being inhibited by MCFA (Hristov et al. 2009).
Supplementation of COD reduced NH3 concentrations which may have been a
consequence of the rumen protozoa population being reduced by 81% in COD
supplemented refaunated heifers that could indirectly affect deamination activity of
rumen the rumen biota. This reduced population of protozoa may allow a compensatory
increase in bacterial population, leading to more NH3 utilisation by bacteria for protein
synthesis (Machmüller and Kreuzer 1999) which, together with less degradation of
dietary protein in the rumen (Jouany and Ushida 1999; Zeitz et al. 2012) would both
reduce NH3 accumulation. Sutton et al. (1983) found greater microbial synthesis in
sheep supplemented with CO, however, the present study and a study of Machmüller
and Kreuzer (1999) showed that microbial protein outflow was not increased by CO
supplementation. This is because CO not only supresses the protozoal population but
Chapter 6: Defaunation and oil effects on rumen fermentation
147
also suppresses rumen bacteria (Dohme et al. 1999), which probably constrains
microbial synthesis in the rumen.
Consistent with other studies (Sutton et al. 1983; Machmüller and Kreuzer 1999; Lovett
et al. 2003; Hollmann et al. 2012), this study found 26% reduction in DMI in heifers
given COD, thus the reduced CH4 emissions were associated with an undesirable
reduction in feed intake. This reduction in DMI was probably due to high level of lauric
acid (42%) and myristic acid (15%) in COD as lauric and myristic acids depress feed
intake (Dohme et al. 2001; Hristov et al. 2011). The effect of COD on DMD, digesta
kinetics and ADG was inconsistent with literature (Sutton et al. 1983; Machmüller and
Kreuzer 1999; Hollmann et al. 2012) who showed CO reduced ruminal fibre
degradation and NDF digestion, which was probably due to the inhibited fibrolytic
bacteria by dietary fat (Nagaraja et al. 1997). However, Machmüller et al. (2003) fed
sheep 50g CO/kg dry matter and showed no adverse effects on the total tract dry matter
digestion or energy retention. Jordan et al. (2006) also showed that feeding 250g/d of
refined CO to beef heifers maintained intake and improved animal performance.
Supplementation with COD in this study reduced DMI and NH3 concentration, but total
VFA concentration and ADG were not affected, which probably reflects the COD
increasing the average energy content of the diet consumed.
Chapter 6: Defaunation and oil effects on rumen fermentation
148
6.5 Conclusion
This experiment confirms that defaunation of the rumen and supplementation with COD
independently reduces CH4 emissions of cattle. The effects of defaunation in
combination with COD in reducing DMP and MY were additive. However, the reduced
emissions achieved by defaunation and supplementation with COD occurred without
altering total VFA, the proportions of propionate and acetate, gut digesta kinetics or
ADG. Further work is required to fully understand the effects of defaunation and COD
supplementation on ruminal fermentation, gut digesta kinetics and CH4 emissions.
Chapter 6: Defaunation and oil effects on rumen fermentation
149
Higher Degree Research Thesis by Publication
University of New England
Statement of Originality
(To appear at the end of each thesis chapter submitted as an article/paper)
We, the PhD candidate and the candidate’s Principal Supervisor, certify that the
following text, figures and diagrams are the candidate’s original work.
Type of work
Paper numbers
Journal article 125-148
Name of Candidate: Son Hung Nguyen
Name/title of Principal Supervisor: Prof. Roger Stephen Hegarty
__________________ 14 June 2016
Candidate Date
_________________ 14 June 2016
Principal Supervisor Date
Chapter 6: Defaunation and oil effects on rumen fermentation
150
Higher Degree Research Thesis by Publication
University of New England
STATEMENT OF AUTHORS’ CONTRIBUTION
(To appear at the end of each thesis chapter submitted as an article/paper)
We, the PhD candidate and the candidate’s Principal Supervisor, certify that all co-authors
have consented to their work being included in the thesis and they have accepted the
candidate’s contribution as indicated in the Statement of Originality.
Author’s Name (please print clearly) % of contribution
Candidate Son Hung Nguyen
85%
Other Authors Roger Stephen Hegarty
15%
Name of Candidate: Son Hung Nguyen
Name/title of Principal Supervisor: Prof. Roger Stephen Hegarty
__________________ 14 June 2016
Candidate Date
_________________ 14 June 2016
Principal Supervisor Date
Chapter 7: Protozoal distribution in the foregut of cattle
151
Chapter 7
Distribution of ciliate protozoa populations in the
rumen, reticulum, and omasum of Angus heifers
S. H. Nguyena, b
and R. S. Hegartya
a School of Environmental and Rural Science, University of New England, Armidale,
NSW 2351, Australia
b National Institute of Animal Sciences, Thuy Phuong, Tu Liem, Hanoi, Vietnam
Journal of Animal Physiology and Animal Nutrition (under revision)
Chapter 7: Protozoal distribution in the foregut of cattle
152
Abstract
Ruminal, reticular and omasal contents and tissues were collected from Angus heifers (n
= 8) at slaughter to determine the total protozoal population and its distribution within
different parts of cattle foregut. The majority of protozoa at slaughter (99%) were
present in free digesta not adhering to the gut wall (1.0%). The populations in the
reticular and omasal digesta, while much smaller than (~5% of) that in the rumen, were
of similar size to each other. The omasum surface, however, provides sequestration for
a similar number of protozoa than does the entire rumen surface, indicating that the
omasum may be an important reservoir for protozoa, especially entodiniomorphs. This
is a likely reason why there are so few reports of cattle sustaining a protozoa-free rumen
for a prolonged period.
Keywords: ciliate protozoa, foregut, cattle
7.1 Introduction
Removal of protozoa from the rumen (defaunation) increases bacterial biomass and
increases flow of protein into the duodenum (Bird and Leng 1978; Jouany 1996), which
is associated with a 9-35% increase in growth rate of defaunated relative to faunated
ruminants (Bird 1989). Defaunation can also decrease enteric methane production
(Kreuzer et al. 1986; Hegarty 1999; Morgavi et al. 2008) by eliminating methanogens
that exist as endo- and ecto-symbionts with ciliate protozoa (Finlay et al. 1994) and by
changing the molar proportions of VFA to a greater proportion of propionate and lesser
proportion of butyrate (Eugène et al. 2004a). However, available techniques to
completely remove protozoa are severe, possibly due to the difficulty in eliminating all
Chapter 7: Protozoal distribution in the foregut of cattle
153
omasal protozoa, which are thought to migrate back into the rumen after ruminal
defaunation is complete (Towne and Nagaraja 1990). Having shown there was merit in
defaunation of cattle (Chapter 6) but great difficulty which was suggested as being
caused by survival of an omasal population which resupplied the rumen, this study
aimed to quantify resident ciliate protozoa populations both in the digesta and on the
surface of the reticulum, rumen and omasum of Angus cattle.
7.2 Materials and methods
7.2.1 Animals, feed and sampling
All protocols for treatment and care of the cattle were approved by the University of
New England Animal Ethics Committee (AEC 14-117). Angus heifers (n = 8; 226 ±
11.8 kg; 8 months of age) were offered 12 kg/head/day of a chaffed lucerne cereal hay
mix (9.7 MJ ME/kg DM and 14.3% CP, Table 7.1) with the feed offered twice daily in
two equal portions.
Samples of rumen fluid were collected every 2 weeks from each heifer before feeding
using oesophageal intubation for protozoal enumeration from day 0 to day 42. On day
45, heifers were transported by truck 30 min to a local abattoir and killed the same
morning with no feed being offered. Immediately after slaughter and evisceration, the
reticulum and omasum were located and tied off to avoid flow of digesta within the
foregut. The complete reticulum, rumen and omasum with and without digesta were
weighed individually to determine weights of digesta contained, and tissue weight of
each organ. Digesta from each organ was thoroughly mixed and approximately 20 g
sub-samples were collected in 25 mL open ended syringes with the tops cut off. The
Chapter 7: Protozoal distribution in the foregut of cattle
154
liquid was then squeezed out by placing a doubled layer of cheese-cloth over the open
end then pushing the plunger into the barrel of the syringe. The pH of the resulting
strained liquid (liquid fraction) was immediately measured using a portable pH meter
(Orion 230 Aplus, Thermo scientific, Beverly, MA, USA) and then the liquid samples
were preserved in pre-weighed containers containing formaldehyde-saline (4% formalin
v/v; 0.9% NaCl w/v). The particulate digesta retained on the cheese-cloth (solid
fraction) was also preserved in pre-weighed containers containing formaldehyde-saline.
The containers were later re-weighed to determine weights of liquid and solid samples.
Gut tissue samples were cut from each organ where the locations were identically
located in each animal. Samples of gut tissues were fixed on plastic boards (3.5×4.5 cm)
and then the samples were gently rinsed in clean water to wash off the trapped digesta
before being preserved in pre-weighed containers with 10% (v/v) formalin to enable
adherent protozoa to be counted. The containers were later re-weighed to determine the
weight of gut tissue preserved.
Chapter 7: Protozoal distribution in the foregut of cattle
155
Table 7.1 Chemical composition of the lucerne cereal hay mix (g/100g dry matter).
Component Lucerne cereal hay mix
Dry matter (in feed as-fed) 88.7
Dry matter digestibility 69
Digestible organic matter 67
Organic matter 90
Neutral detergent fibre 42
Acid detergent fibre 31
Crude protein 14.3
Crude fat 1.4
Metabolisable energy (MJ/kg DM) 9.7
7.2.2 Sample processing and protozoal enumeration
A subsample (1.0 mL) of preserved liquid fraction was pipetted into a test tube. Two
drops (0.05 mL) of brilliant green (2.0 g of brilliant green dye and 2.0 mL of glacial
acetic acid diluted to 100 mL with distilled water) were added (Dehority 1984). The
contents were mixed and allowed to stand overnight before counting of protozoal cells
by microscopy.
A portion of each preserved sample of the digesta ‘solid’ fraction was further diluted
with formaldehyde-saline, vortexed and then sonicated for 5 min to remove adherent
protozoa. The homogenized preserved ‘solids’ samples were squeezed out through a
doubled layer of cheese-cloth to separate liquid and solid fractions. A subsample (1.0
Chapter 7: Protozoal distribution in the foregut of cattle
156
mL) of liquid was pipetted into a test tube to be stained with brilliant green. The solid
content was placed in a 25 mL beaker to be thoroughly mixed and then 1.0 g of
subsample was placed in a test tube. Three drops (0.075 mL) of brilliant green were
added and vortexed to ensure thorough mixing. The contents were allowed to stand
overnight. The preserved gut tissue samples were placed in a 50 mL beaker, thoroughly
mixed and sonicated for 15 min to release adherent protozoa from the tissue. A 1.0 mL
sample of sonicated preserved liquid fraction was pipetted into a test tube to be stained
with brilliant green and allowed to stand overnight.
After staining, a portion of each stained sample was diluted with 30% glycerol, resulting
in 1:20 dilution of the original sample. Protozoa were counted using a Fuchs–Rosenthal
optic counting chamber (0.0625 mm2 and 0.2 mm of depth). The protozoa were
differentiated into large (>100 µm) and small (<100 µm) holotrich and entodiniomorph
groupings. Total protozoal populations in, or on an organ’s surface, were estimated as
the product of weight of digesta (or weight of tissue) times the protozoa/g of digesta or
protozoa/g of rinsed gut tissue for that organ.
7.2.3 Statistical analyses
Data were subject to analysis of variance (PROC GLM) using SAS 9.0 (SAS Institute,
Cary, NC). Protozoa counts were log-transformed to meet homogeneity of variance and
normal distribution criteria using PROC UNIVARIATE before statistical analysis.
Least significant differences were used for means separation (P < 0.05).
Chapter 7: Protozoal distribution in the foregut of cattle
157
7.3 Results
Rumen protozoal concentrations (cells/mL) monitored over 42 days prior to slaughter
(Figure 7.1) showed protozoal concentrations had increased after initial introduction to
the mixed lucerne and cereal chaff diet (a 48% increase over first 14 days; 1.39×105 v
2.68×105; P < 0.05). There were no significant differences in protozoal concentrations
from day 14 to day 42 (P > 0.05), indicating that concentrations of rumen protozoa were
stable in the weeks leading up to slaughter. Protozoal concentrations from the strained
rumen fluid at slaughter on day 45 were similar to that of samples collected on day 0,
14, 28 and 42 by oesophageal intubation (Figure 7.1; P > 0.05). Small entodiniomorphs
were dominant in the rumen fluid, ranging from 47 to 65% of the total protozoa (Figure
7.1).
The pH of digesta did not differ between reticulum, rumen and omasum. The rumen had
the largest mass of digesta of all the forestomachs (Table 7.2). The total number of
protozoa in the rumen was higher than in the reticulum and omasum. Although the
quantity of digesta in the reticulum was smaller than in the omasum, the total number of
protozoa was similar in both reticulum and omasum.
Chapter 7: Protozoal distribution in the foregut of cattle
159
Table 7.2 Ciliate protozoa in reticular, ruminal and omasal contents and adhering to the
gut tissues of Angus heifers.
Parameter
Mean (n=8) Pooled
s.e P-value
Reticulum Rumen Omasum
pH 6.29 6.38 6.28 0.13 0.83
Digesta weight in grams 905c
34,113a
4,540b
678.3 <0.001
Total protozoa (×106) 162.4
b 7,480.1
a 208.5
b 1.32 0.01
Large holotrichs 29.81b
594.67a
14.82b
1.46 <0.001
Small holotrichs 60.40b
775.11a
15.67c
1.44 <0.001
Large entodiniomorphs 19.43c
1,966.5a
46.81b
1.28 <0.001
Small entodiniomorphs 37.71b
3.677.5a
111.05b
1.45 <0.001
Gut tissue weight in grams 1,111.3c
6,446.3a
4,901.3b
245.3 <0.001
Total protozoa (×105) 0.65
b 3.31
ab 3.74
a 0.28 0.003
Large holotrichs 0.15b
1.01a
- 0.49 0.01
Small holotrichs 0.33b
2.38a
- 0.36 0.003
Large entodiniomorphs 0.12b
1.62ab
2.27a
0.26 <0.001
Small entodiniomorphs 0.11b
0.84ab
2.31a
0.28 <0.001
Different superscripts indicate significant difference within rows
7.4 Discussion
Rumen ciliate protozoa often represent approximately 1×106 cells per mL in rumen
contents (Dehority 2003), but protozoal concentrations vary among animals and are
dependent on many factors such as ruminant species, geographical location (Akbar et al.
2009), diet (Whitelaw et al. 1984), frequency of feeding (Williams 1986) and rumen pH
(Clarke 1977). Monitoring protozoal populations in rumen fluid in this study showed
protozoal concentrations after 14 days of acclimatization period of changing to the diet
of lucerne cereal hay mix were significantly higher than on the first day they started on
the diet. The protozoal populations in rumen fluid after straining through a double layer
Chapter 7: Protozoal distribution in the foregut of cattle
160
of cheese-cloth to remove large plant fibres were similar to that of samples collected
from oesophageal intubation (Figure 7.1; P > 0.05), indicating that experiments
involving sampling rumen fluid from oesophageal intubation for protozoal enumeration
may offer an accurate estimate of protozoal populations being free in the liquid fraction
of the rumen contents. This normal method of enumeration does not account for the
protozoa adhering to plant particles (Bauchop and Clarke 1976) and these authors found
high concentrations of rumen protozoa attaching on the damaged surface of plant
fragments, between layers of plant cells and among vessel elements with the protozoa
being identified as the entodiniomorphs: Epidinium ecaudatum, Eudiplodinium spp.,
Diplodium spp. and the holotrich Dasytricha spp. In addition, Czerkawski (1987)
showed that protozoal populations in the rumen are divided into two distinct
compartments: those freely suspended in the liquid phase, and those protozoa
adhering to the fibrous mass of digesta. Hook et al. (2012) also reported the majority
(63 - 90%) of rumen protozoa existed in the attached phase, either in the feed particles
or in the rumen wall. Leng et al. (1981) who injected radioactivity-labelled protozoa in
the rumen estimated between 60 and 90% of the total protozoa were available within the
rumen to be sampled, but only 5 to 20% of the protozoal population was present in the
fluid fraction.
The pH did not differ among reticular, ruminal and omasal contents and agreed with pH
ranges found in previous studies (Prins et al. 1972; Towne and Nagaraja 1990). Rumen
protozoa are significantly affected by the environment’s acidity or alkalinity, with the
protozoa unable to survive if rumen pH is above 7.8 or below 5.0 (Clarke 1977).
Mackie et al. (1978) reported that protozoal numbers decreased by 50-80% if rumen pH
Chapter 7: Protozoal distribution in the foregut of cattle
161
was below 5.4. The pH of ruminal or omasal contents in the present study were near
neutral as the host animals were fed a roughage diet, meaning the omasum was
therefore a suitable sequestration site for protozoa. Although the protozoal numbers in
omasal contents were significantly fewer than in ruminal contents, which agreed with
Weller and Pilgrim (1974) and Michalowski et al. (1986), similar numbers of protozoa
were adhering to the ruminal and omasal walls.
Ciliate protozoa contribute disproportionately little to the nitrogen nutrition of
ruminants with protozoal nitrogen being up to 53.4% of total microbial biomass in the
rumen (Michałowski 1979) but only 20% of total microbial nitrogen entering to the
duodenum (Jouany et al. 1988). The smaller protozoal biomass in the duodenum of the
ruminants could reflect 65% - 74% of protozoa die and are degraded in the rumen of
sheep or cattle, respectively (Leng 1982; Ffoulkes and Leng 1988), suggesting that only
24% - 35% of rumen protozoa enter to the lower digestive tract, while the majority of
rumen protozoa are retained and lyse within the rumen. Further, the protozoal biomass
leaving the rumen is greater than that arriving in the duodenum because of some rumen
protozoa being trapped in the omasal leaves (Czerkawski 1987). The relatively high
numbers of protozoa found in the omasum in this study may explain that the lower
protozoal contribution to the total microbial nitrogen outflow in the duodenum is partly
due to high numbers of protozoa being retained in the omasum of ruminants.
Elimination of rumen protozoa increases growth rate of ruminants (Bird and Leng 1978;
Bird et al. 1979; Eugène et al. 2004a) especially when feed is deficient in protein rather
than energy content. This leads to some circumstances in which rumen protozoa may
Chapter 7: Protozoal distribution in the foregut of cattle
162
limit animal productivity. However, existing procedures to eliminate ciliate protozoa
involve dosing ruminal contents with antiprotozoal detergents (Abou Akkada et al.
1968; Orpin 1977; Santra et al. 2007a; Bird et al. 2008; Hegarty et al. 2008), but these
methods of defaunation are not always successful. In sheep, defaunation had been
successfully with sodium 1-(2-sulfonatooxyethoxy) dodecane (Bird et al. 2008; Hegarty
et al. 2008) or sodium lauryl sulfate (Santra et al. 2007a), but chemical defaunating
detergents are not often reported successful in rendering cattle free of ciliate protozoa
for prolonged periods (Bird and Leng 1978). Towne and Nagaraja (1990) completely
eliminated rumen protozoa of Holstein steers by emptying ruminal contents, flushing
the omasum and spraying 1 L of dioctyl sodium sulfosuccinate solution on rumino-
reticulum walls and on the reticulo-omasal orifice. The authors found live protozoa in
the rumen contents a day after the treatment.
The omasum, which is connecting the reticulorumen to the abomasum, transfers digesta
from the reticulum into the abomasum (Van Soest 1994). The flow of digesta from the
reticulum occurs following the omasal canal contractions, but occasionally backflow of
large volumes of digesta from the omasum to the reticulum occurs when the omasal
body contracts during the closure of omaso-abomasal orifice (Stevens et al. 1960).
Therefore, Towne and Nagaraja (1990) claimed that the backflow of omasal contents
containing residual of omasal protozoa re-inoculated the defaunated rumen of steers.
That could explain why defaunation of cattle is difficult as residual omasal protozoa
may be responsible for the reappearance of rumen protozoa after ruminal defaunation is
complete.
Chapter 7: Protozoal distribution in the foregut of cattle
163
7.5 Conclusion
The relatively large total populations of ciliate protozoa in the digesta and on the rumen
wall of cattle indicate that the total populations of protozoa in the rumen cannot be
simply counted by the conventional enumeration of fluid samples, although
concentrations of rumen protozoa in the rumen fluid were similar in samples collected
by oesophageal intubation and by slaughter. This study confirms there is a small but
significant reservoir of protozoa in the omasum of cattle and it is proposed their
backflow into the rumen contributes to reducing the effectiveness of chemical
defaunation of the rumen. Techniques that completely eliminate protozoa from the
omasum as well as the rumen of cattle will be required if the advantages of increasing
the microbial nitrogen outflow and efficiency of feed utilisation associated with
elimination of rumen protozoa are to be realised in the cattle industries.
Chapter 7: Protozoal distribution in the foregut of cattle
164
Higher Degree Research Thesis by Publication
University of New England
Statement of Originality
(To appear at the end of each thesis chapter submitted as an article/paper)
We, the PhD candidate and the candidate’s Principal Supervisor, certify that the
following text, figures and diagrams are the candidate’s original work.
Type of work
Paper numbers
Journal article 151-163
Name of Candidate: Son Hung Nguyen
Name/title of Principal Supervisor: Prof. Roger Stephen Hegarty
__________________ 14 June 2016
Candidate Date
_________________ 14 June 2016
Principal Supervisor Date
Chapter 7: Protozoal distribution in the foregut of cattle
165
Higher Degree Research Thesis by Publication
University of New England
STATEMENT OF AUTHORS’ CONTRIBUTION
(To appear at the end of each thesis chapter submitted as an article/paper)
We, the PhD candidate and the candidate’s Principal Supervisor, certify that all co-authors
have consented to their work being included in the thesis and they have accepted the
candidate’s contribution as indicated in the Statement of Originality.
Author’s Name (please print clearly) % of contribution
Candidate Son Hung Nguyen
85%
Other Authors Roger Stephen Hegarty
15%
Name of Candidate: Son Hung Nguyen
Name/title of Principal Supervisor: Prof. Roger Stephen Hegarty
__________________ 14 June 2016
Candidate Date
_________________ 14 June 2016
Principal Supervisor Date
Chapter 8: General discussion
167
Chapter 8
General discussion
8.1 Introduction
Research on rumen protozoa has largely focused on their classification, effects on
ruminal fermentation and protein yields (Williams and Coleman 1992), but as enteric
CH4 production from ruminants is the largest agricultural emission contributing to
global warming, the role of rumen protozoa in moderating rumen methanogensis has
received increasing attention (Newbold et al. 2015). Dietary strategies such as feeding
of oils, saponins and NO3 have also been examined to manipulate ruminal metabolism
to reduce CH4 production. Part of the action of some of these dietary interventions is
through affecting rumen protozoal populations (Newbold et al. 2015), making it
difficult to assess whether CH4 mitigation is a direct effect of the additives or an indirect
effect on protozoal density or both. Studies contrasting defaunated and refaunated
animals, therefore, offer an opportunity to understand the contribution of protozoa to
ruminal metabolism without confounding by diet ingredients, but even these have built
an unclear understanding in the published literature of the role of protozoa (Williams
and Coleman 1992). The major aim of this thesis was to understand the CH4 production
and animal productivity associated with defaunation and whether additive changes in
CH4 mitigation and productivity were possible by using defaunation and dietary
additives together. The studies also sought to extend the applicability of defaunation by
Chapter 8: General discussion
168
measuring emissions of defaunated sheep while grazing and by investigating protozoal
retention in the reticulum and omasum in order to facilitate future defaunation of cattle.
8.2 Protozoal impacts on the rumen and its fermentation and
methane production
Perhaps the greatest consequence of defaunation for the rumen ecosystem and
fermentation chemistry is a reduced predation of bacteria and an increased bacterial
population. This increases microbial protein outflow and in turn increases animal
productivity, especially where low protein diets are limiting animal production
(Williams and Coleman 1992; Newbold et al. 2015). A lower concentration of NH3 in
the defaunated rumen was the most consistent effect of defaunation reported in this
thesis and in the literature (Table 8.1). A decrease in rumen NH3 level is a consequence
of the absence of protozoa reducing both bacterial predation and the degradation of
feed-protein in the rumen (Williams and Coleman 1992). The higher nitrogen flow into
the duodeum results from an increase in feed nitrogen and microbial nitrogen flow
(Ushida and Jouany 1990), leading to increased supply of amino acids to the host.
Chapter 8: General discussion
169
Table 8.1 Rumen metabolite concentration and methane production in the rumen fluid
of defaunated animals normalized relative to those in faunated animals (1.00). Data are
from experiments in this thesis and from published reviews.
Total
VFA Acetate Propionate Butyrate
Acetate/
Propionate NH3
Methane
production
Chapter 2 1.04 1.08*
0.89*
0.76*
1.21 0.74*
0.93*
Chapter 3†
0.91 0.96*
1.01 1.25*
0.94 0.73*
0.97
Chapter 4 0.84*
1.05*
0.85*
0.93 1.23*
0.66*
0.57*
Chapter 5‡
0.79
1.02 1.04 0.84 0.98 0.76*
0.62*
Chapter 6 1.16 1.01 1.10 0.89 0.91 0.61*
0.90*
Thesis mean 0.95 1.02 0.98 0.93 1.05 0.70 0.80
Published review
Jouany et al. (1988) 0.94 0.98 1.32 0.94 0.90 0.76
Hegarty (1999) 0.86 1.23 1.08 0.70 0.87
Eugène et al.
(2004a) 0.96 0.98 1.14 0.87 0.86 0.70
Newbold et al.
(2015) 0.95 1.03 1.00 0.78 1.03 0.74 0.89
* Significant effect of defaunation (P < 0.05);
† Experiment conducted under grazing environment;
‡ In
vitro experiment.
The extent of rumen fermentation and the balance of energy yielding substrates for the
host are also important. The reduced total VFA concentration apparent in data averaged
across studies in this thesis was consistent with the literature (Table 8.1). The lower
total VFA concentration in the defaunated rumen could be due to a reduced rate of VFA
production (Chapter 4) and/or a larger rumen volume in which the fermentation was
occuring (Chapter 2). The experimental evidence presented in this thesis, however,
confirms that protozoa effects on the molar proportions of VFA are not consistent. An
increased molar proportion of propionate in the defaunated rumen was evident in many
existing reviews (Jouany et al. 1988; Hegarty 1999; Eugène et al. 2004a), but our
Chapter 8: General discussion
170
results showed defaunation consistantly increased the molar proportion of acetate in
agreement with Newbold et al. (2015) and subtantially reduced the molar proportion of
butyrate, but did not significantly increase the molar proportion of propionate.
The results presented in this thesis support a conclusion that defaunation reduces CH4
production both in vitro and in vivo (Table 8.1) and provided the first measurement of
CH4 production of defaunated ruminants while grazing. However, the percentage
emission reduction was variable and mechanisms by which CH4 emissions are reduced
by defaunation are not clear. Hegarty (1999) proposed four possible mechanisms by
which defaunation induces a lower CH4 emissions, being; (1) reduced DM fermentation
in the rumen (2) decreased endosymbiotic methanogens associated with rumen protozoa
(3) modified ruminal VFA profile with increased molar proportion of propionate and
decreased availability of H2 (4) increased oxygen pressure in rumen fluid. Nevertheless,
the experiments in this thesis recognise the complexity of the effect of defaunation on
the ecosystem of the rumen, revealing a reduced CH4 emissions can occur without an
increased proportion of propionate.
By removing rumen protozoa, defaunation must eliminate the ecto- and endo-symbiotic
habitats for physically associated methanogens (Finlay et al. 1994; Tokura et al. 1997;
Kumar et al. 2013). However, as CH4 emissions are not always decreased by
defaunation (Kumar et al. 2013), alternative methanogen populations may arise and
replace those of the protozoa-associated methanogens (Morgavi et al. 2012). The
changes in the methanogenic community following defaunation are inconsistent among
studies (McAllister and Newbold 2008; Mosoni et al. 2011; Morgavi et al. 2012; Kumar
Chapter 8: General discussion
171
et al. 2013). Reduced CH4 emissions following defaunation in these studies may be due
to reducing the most active CH4 methanogens in the rumen and the substitution of other
methanogenic populations which are less able to utilise H2 to produce CH4. Another
possibility is that in the absence of protozoa other populations of microbes establish or
increase in the rumen that may enable alternative sinks for H2 that have a higher affinity
for H2 than do methanogens. While our circumstantial data supports this with more
acetate and less CH4 as may be expected if reductive acetogenesis was active (Chapter
2, 4 and 6), this hypothesis was not tested and needs future investigation as it is
recognised that reductive acetogenesis is not a normal ruminal reaction.
The results from Chapter 2 showed that the absence of rumen protozoa tended to
increase reticulo-rumen weight and significantly increase the ratio of reticulo-rumen to
whole body weight. The increased weight of rumen contents are often seen after
defaunation due to a longer particle retention of rumen digesta (Chapter 4) associated
with the rumen fill effect of reducing OM (Eugène et al. 2004a) and/or whole tract DM
digestibility (Chapter 4). This physical change in the rumen size following defaunation,
however, is not always observed (Williams and Coleman 1992).
Although the changes in rumen characteristics and VFA molar proportions are not
always consistent following defaunation, this thesis has clearly demonstrated the
consistent influence of rumen protozoa on NH3 concentration. The reduced ruminal
catabolism of engulfed feed-protein and bacteria as well as reduced CH4 emissions are
advantages of defaunation. These advantages would favour an enhanced efficiency of
Chapter 8: General discussion
172
nutrient utilisation and production of ruminants, especially those with a high amino acid
requirement and/or when consuming feeds of very low protein content.
8.3 Growth and productivity of defaunated ruminants
The demand for products from livestock, especially in the tropics where forage is often
deficient in protein content, is increasing. The requirement to produce at least 70% more
food in order to feed 9 billion people by 2050 (World Bank 2008) is a major challenge
to animal production. The world livestock population has surged in many developing
countries in response to this rapid growing demand for livestock products (FAO 2006).
In Asia, the majority of ruminants are fed protein deficient diets from locally produced
and available by-products due to an increasing competition for feed between human
consumption and monogastric livestock demands (Devendra and Leng 2011).
Defaunation of the rumen offers an opportunity to optimise productivity of ruminants in
such protein-scarce environments. This thesis demonstrated a consistent effect of
defaunation to increase microbial protein outflow which increased protein supply to the
host for liveweight gain (Table 8.2). Although a positive response to defaunation in
animal performance is not always significant in the literature, there are no apparent
negative effects. A small decrease in DM digestibility due to defaunation in this thesis
and in the review was not associated with reduced animal growth, suggesting that
greater microbial protein supply may lead to higher feed conversion efficiency in
defaunated ruminants. Indeed, defaunation has increased feed conversion efficiency due
to a greater efficiency of nutrient utilisation for absorption compared to conventional
animals (Newbold et al. 2015). These positive effects of defaunation on animal growth
Chapter 8: General discussion
173
are often seen with poor quality roughage diets that are low in fermentable carbohydrate
and rumen degradable nitrogen for the growth of rumen microbes (Chapter 4).
Table 8.2 Dry matter intake, digestibility, microbial protein outflow, liveweight gain
and wool growth of defaunated ruminants normalized relative to those of faunated
ruminants (1.00). Data are from experiments in this thesis and from published reviews.
DM
intake
DM
digestibility
Microbial
protein
outflow
Liveweight
gain
Wool
growth
Wool
fibre
diameter
Greasy
fleece
weight
Chapter 2 0.98*
0.97 1.17 1.04
Chapter 3†
1.05 1.03 0.97 1.03 1.02
Chapter 4 0.94 0.94*
1.03 1.11 0.95 1.01
Chapter 6 0.98 1.03 1.29*
1.56
Thesis mean 0.99 0.98 1.16 1.18 0.94 1.02 1.02
Published review
Jouany et al.
(1988) 1.04 0.94 1.56 1.02
Eugène et al.
(2004a) 1.01 0.98 1.12 1.11
1.14
Newbold et al.
(2015) 0.98 0.96 1.30 1.09 1.01
* Significant effect of the defaunation (P < 0.05); † Experiment conducted under grazing environment
The experiments reported in Chapter 3 and 4 showed that wool production was not
positively increased by defaunation, but wool fibre diameter was slightly increased
(Table 8.2). Previous studies have shown positive effects of defaunation on wool
growth (Eugène et al. 2004a) and longer experimental periods or higher statistical
power in the current experiments may have likely shown similar results. This thesis also
showed a greater liveweight gain response in defaunated cattle (Chapter 6) associated
with a greater quantity of microbial protein which flows from the bovine rumen is
available for muscle growth. This suggests that defaunation of beef cattle would have
Chapter 8: General discussion
174
both commercial and environmental benefits, and for these reasons, the study of
protozoa in the forestomach of cattle was undertaken (Chapter 7; section 8.5).
8.4 Defaunation and dietary oil or nitrate as complementary
mitigation strategies
Many of the leading feed additives showing efficacy in CH4 mitigation are likely to be
commercially constrained due to either toxicity risk (NO3; Leng and Preston (2010)) or
suppression of DM intake and DM digestibility (eg. oils; Patra (2013)), that limits their
inclusion in the diet. Recently, Hristov et al. (2015) reported an active compound of 3-
nitrooxypropanol (3NOP) that persistently reduces CH4 emissions in dairy cattle. Thus,
there is interest in combining mitigation strategies to achieve greater total CH4
mitigation. Our studies, like those of Guyader et al. (2015) and Troy et al. (2015)
combined defaunation with supplements of dietary NO3 (Chapter 4) and coconut oil
(Chapter 6) to supplement animals with NPN (NO3) or energy (coconut oil) which may
be both advantageous to animal eating low quality forage. Results from Chapter 4
showed that supplementation with NO3 increased DM intake and DM digestibility,
rumen fermentation and ADG while supplementation of coconut oil reported in Chapter
6 did not improve rumen fermentation or animal productivity, largely as a result of
suppressing DM intake.
Although supplementation of NO3 or coconut oil independently reduced CH4
production, combining these with defaunation resulted in different outcomes for CH4
mitigation. The results presented in Chapter 4 showed for the first time that defaunation
and NO3 positively interacted to reduce CH4 yield and that NO3 in combination with
Chapter 8: General discussion
175
defaunation additively increased DM intake and decreased CH4 production. Nitrate is
known to serve as a H2 sink that could favour NH3 formation over CH4 formation in the
rumen, and it also indirectly decreases the methanogen population (van Zijderveld et al.
2011). Combining defaunation with NO3 could be further affecting methanogens to
reduce CH4 production in an additive manner compared to NO3 treatment alone.
The lack of interaction between defaunation and coconut oil reported in Chapter 6
supported the hypothesis that both treatments would additively deliver greater
mitigation of CH4 emissions compared to defaunation or oil treatment alone.
Combination of defaunation and coconut oil showed the reduced rumen NH3
concentration and microbial protein outflow from defaunated rumen. This is because
coconut oil not only suppresses the rumen protozoal population which indirectly
decreases CH4 production, but also suppresses the rumen bacteria (Dohme et al. 1999),
which constrains microbial synthesis in the rumen. The non-significant interaction
between defaunation and coconut oil on CH4 mitigation reported in Chapter 6 confirms
a previous study by Machmüller et al. (2003) who found no interaction for methane
production, but a higher density of methanogens in the rumen fluid of defaunated sheep
when fed coconut oil. Therefore, CH4 mitigation was not found to be further enhanced
by supplementing defaunated animals with oils.
Studies from this thesis confirm that supplementation with NO3 as a NPN source (but
not coconut oil) enhanced defaunated animal productivity and additively reduced CH4
emissions.
Chapter 8: General discussion
176
8.5 Challenges to applying defaunation in commercial practice
Despite the fact that elimination of rumen protozoa has shown potentially positive
impacts on improving animal productivity and reducing enteric CH4 emissions from
ruminants, there are no defaunation methods that are safe, effective and practically
applicable for commercial enterprises. For research purposes, Jouany et al. (1988) had
reviewed different techniques used to defaunate the rumen. However, these techniques
are not always successful in rendering ruminants, especially cattle, free from protozoa
for a prolonged period of time.
This thesis further advances the technique of Bird and Light (2013) to defaunate the
ovine rumen by feeding animals with coconut oil distillate rich in lauric acid to suppress
rumen protozoa for at least 7 days before three days of orally dosing with sodium 1-(2-
sulfonatooxyethoxy) dodecane. However, the dosing procedure may still require
repeating for permanent defaunation. This defaunation technique suppresses animal
intake for an average of 10 days, but it is still not clear what period of time is required
for the microbial ecosystem to fully stabilise.
Defaunation of cattle in this study was not easy achieved as entodiniomorph protozoa
were visually observed 4 weeks after the experiment finished, so the cattle were visually
free from ciliate protozoa for only 12 weeks. Defaunation of cattle, therefore, is more
difficult than for sheep and this is thought to be attributable to different anatomical
structures between the ovine and bovine rumen and omasum that restrict the exposure of
protozoa to the defaunating chemicals (Towne and Nagaraja 1990). The finding of a
small but significant protozoal population in the omasum (contents and adhering to the
Chapter 8: General discussion
177
wall, Chapter 7) supports the hypothesis of Towne and Nagaraja (1990) that protozoa
residing in the omasum could provide a reservoir of organisms that re-infect the rumen
after defaunation treatments have ceased. This presents a commercial challenge to use
of oral or in-feed defaunating treatments and suggests an antiprotozoal compound that is
carried in blood before diffusing into the gastrointestinal tract may be required to
successfully defaunate cattle commercially. Since no such compounds are available, this
is a major constraint to defaunation of bovines in the tropical regions where protein
deficiency is limiting productivity of ruminants.
8.6 Conclusion
Studies in this thesis confirm for the first time that sheep without protozoa have a lower
CH4 yield while grazing than do faunated sheep, although the mitigation is moderate
and only statistically different with P < 0.1. Across all housed animal and grazing
studies the effects on rumen fermentation were mixed, as evident in existing literature.
Consequently, defaunation cannot be relied upon to change fermentation patterns in a
predictable way. The reliability of changes in ruminal and animal nitrogen metabolism
in the thesis and wider literature are, however, consistent and of sufficient importance to
motivate defaunation of developing anti-protozoal treatments for application in the
tropics where low protein diets are common. It is suggested complete defaunation of
cattle (and presumably buffalo) will require a compound that contacts protozoa by both
direct contact in digesta and by diffusion from the blood to eliminate protozoa
sequestered in the omasum.
Chapter 8: General discussion
178
To provide assurance of CH4 mitigation and enhanced animal productivity it is
recommended that defaunation be combined with other mitigation strategies such as
NO3 feeding where additive effects from NO3 can be expected to not only further reduce
emissions, but also improve animal performance and potentially improve reproductive
outcomes due to improved body weight or condition score of tropical ruminants.
References
179
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